U.S. patent application number 14/561912 was filed with the patent office on 2015-04-02 for use of stem cells to prevent neuronal dieback.
This patent application is currently assigned to ABT Holding Company. The applicant listed for this patent is Sarah A. BUSCH, Kevin P. Horn, Robert W. Mays, Jerry Silver. Invention is credited to Sarah A. BUSCH, Kevin P. Horn, Robert W. Mays, Jerry Silver.
Application Number | 20150093364 14/561912 |
Document ID | / |
Family ID | 41797508 |
Filed Date | 2015-04-02 |
United States Patent
Application |
20150093364 |
Kind Code |
A1 |
BUSCH; Sarah A. ; et
al. |
April 2, 2015 |
USE OF STEM CELLS TO PREVENT NEURONAL DIEBACK
Abstract
The invention is generally directed to treatment of neuronal
injury. In particular, the invention is directed to reducing axonal
retraction ("dieback") that occurs as a result of the interaction
of activated macrophages with dystrophic axons that are produced
during nervous system acute or chronic injury. The invention is
also directed to promoting axonal growth/regeneration. The
invention is specifically directed to using stem cells or their
secreted cellular factors, such as would be produced in conditioned
cell culture medium, to ameliorate or prevent axonal dieback and/or
promote growth/regeneration of axons.
Inventors: |
BUSCH; Sarah A.; (Cleveland,
OH) ; Horn; Kevin P.; (Salt Lake City, UT) ;
Mays; Robert W.; (Shaker Heights, OH) ; Silver;
Jerry; (Bay Village, OH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BUSCH; Sarah A.
Horn; Kevin P.
Mays; Robert W.
Silver; Jerry |
Cleveland
Salt Lake City
Shaker Heights
Bay Village |
OH
UT
OH
OH |
US
US
US
US |
|
|
Assignee: |
ABT Holding Company
Cleveland
OH
Case Western Reserve University
Cleveland
OH
|
Family ID: |
41797508 |
Appl. No.: |
14/561912 |
Filed: |
December 5, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14045582 |
Oct 3, 2013 |
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14561912 |
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13062343 |
Aug 18, 2011 |
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PCT/US09/56046 |
Sep 4, 2009 |
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14045582 |
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61094210 |
Sep 4, 2008 |
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Current U.S.
Class: |
424/93.7 ;
424/577 |
Current CPC
Class: |
A61P 9/00 20180101; A61P
21/02 20180101; A61P 43/00 20180101; A61P 25/16 20180101; A61K
35/12 20130101; A61K 35/28 20130101; A61P 9/10 20180101; A61P 25/28
20180101; C12N 5/0607 20130101; A61K 2035/124 20130101; A61K 35/30
20130101; A61P 25/08 20180101; A61P 25/00 20180101; A61K 35/545
20130101; A61P 21/00 20180101 |
Class at
Publication: |
424/93.7 ;
424/577 |
International
Class: |
A61K 35/28 20060101
A61K035/28 |
Claims
1-13. (canceled)
14. A method for reducing the adhesion of ED-1.sup.+ cells to
dystrophic axons that would result in axonal retraction, said
method comprising administering stern cells, or factors secreted
therefrom, in sufficient proximity to the dystrophic axons and/or
ED-1.sup.+ cells, for time sufficient, and in sufficient amounts to
reduce said adhesion, wherein the stern cell is a human
non-embryonic stem cell that has the ability to differentiate into
cell types of more than one embryonic germ layer and/or express one
or more of oct4, telomerase, rex-1, rox-1, sox-2, and SSEA4.
15. The method of claim 14 wherein reducing said adhesion of
ED-1.sup.+ cells to dystrophic axons in a subject reduces axonal
retraction in said subject.
16. The method of claim 14 wherein reducing said adhesion of
ED-1.sup.+ cells to dystrophic axons reduces axonal retraction in a
subject and reduces neural injury that is associated with said
axonal retraction in said subject.
17. The method of claim 14 wherein reducing said adhesion of
ED-1.sup.+ cells to dystrophic axons promotes axon regeneration in
a subject.
18. The method of claim 14 wherein said ED-1.sup.+ cells are
macrophages and/or microglia.
19. The method of claim 14 wherein the secreted factors are derived
from well culture medium conditioned by culturing the stem cells
therein, the factors being in a pharmaceutically-acceptable
carrier.
20. The method of claim 14 wherein said secreted factors are
administered.
21. The method of claim 20 wherein said secreted factors are in
medium conditioned by culturing the stem cells therein.
22. The method of any of claims 14-21 wherein the stern cells are
derived from bone marrow.
Description
FIELD OF THE INVENTION
[0001] The invention is generally directed to treatment of neuronal
injury. In particular, the invention is directed to reducing axonal
retraction ("dieback") that occurs as a result of the interaction
of activated macrophages with dystrophic axons that are produced
during nervous system acute or chronic injury. The invention is
also directed to promoting axonal growth/regeneration. The
invention is specifically directed to using stem cells or their
secreted cellular factors, such as would he produced in conditioned
cell culture medium, to ameliorate or prevent axonal dieback and/or
promote growth/regeneration of axons.
BACKGROUND OF THE INVENTION
Axonal Retraction
[0002] After spinal cord injury, a glial scar forms that poses a
major impediment to CNS regeneration (Silver and Miller, 2004). In
the region of forming scar tissue, the ends of the regenerating
axons cease extending and become swollen and distorted into various
bizarrely shaped "growth cones" that can remain for years within
axon tracts (Ramon y Cajal, 1928; Li and Raisman, 1995; Houle and
Jin, 2001; Kwon at al., 2002). Injured axons within the CNS
withdraw from the site of axotomy during a period of hours to weeks
after an initial injury. There have been differing reports as to
the nature of axonal retraction, its cause, extent, and timing as
well as discussion of whether it is a passive or active process
(Fayaz and Tator, 2000).
[0003] In vitro Glial Scar Model
[0004] In the region of forming scar tissue, several classes of
growth inhibitory molecules are unregulated, including the family
of extracellular matrix (ECM) molecules known as
chondroitin/keratan sulfate proteoglycans (PGs) (Fitch and Silver,
1997; Morgenstern, at al., 2002; Jones at M., 2003; Tang at al.,
2003). PGs are organized in a crude gradient with the lowest
concentrations in the lesion penumbra and the highest in the
epicenter (Davies et al., 1999; Fitch at al., 1999). The inhibitory
ECM components block the potential of reactive glial cells to
support axonal regeneration via laminin (McKeon at al., 1991).
Microtransplantation experiments show that adult sensory neurons
have a robust capacity for regeneration when placed away from the
lesion. Once the regenerating fibers reach the vicinity of the
injury site, they are capable of struggling into the lesion
penumbra but eventually cease extending and become dystrophic as
they penetrate deeply into areas of highest PG concentration
(Davies et al., 1999; Grimpe and Silver, 2004).
[0005] In vitro glial scar models, that used sharp-edged (i.e.,
stripe) substrate assays to examine the effects of PGs on axons,
induced either growth cone turning or collapse, but not dystrophy
(Snow et al., 1990). However, in a recent model of the glial scar,
a crude gradient of PGs was sufficient to produce dystrophic
endings in regenerating adult axons (Tom at al., 2004). This in
vitro system forces regenerating axons of adult sensory neurons to
cope with a spot gradient of the PG aggrecan mixed with laminin.
Bulbous multivesiculated endings were formed in this glial scar
model. PGs led to growth cone dystrophy and dynamic dystrophic
endings.
Inflammation and Injury in Neuronal Tissue
[0006] The environment of a spinal cord lesion is extremely
complex, Components of the glial scar, such as highly sulfated
proteoglycans, ephs, slits, and myelin membrane fragments, (Silver
and Miller, 2004; Yin and He, 2006; Busch and Silver, 2007) as well
as the process of neuroinflammation (Donnelly and Popovich, 2007)
all contribute to regeneration failure. inflammatory cells
accumulate within the lesion (Fitch at al., 1999), Astrocytes move
away from the center of the lesion, become hypertrophic, and
upregulate production of inhibitory chondroitin sulfate
proteoglycans (CSPGs) that, in turn, cause the formation of
dystrophic endbulbs on the severed fibers (Tom et al., 2004).
Oligodendrocytes within the lesion die, leading to demyelination,
which results in high concentrations of inhibitory myelin breakdown
products (Yiu and He, 2006; Xie and Zheng, 2008).
[0007] While the inhibitory effects of proteoglycans and myelin on
axonal growth were well-established, the role of neuroinflammation
in regeneration and regeneration failure remained highly
controversial (Popovich and Longbrake, 2008). However, studies have
indicated that macrophage infiltration results in increased lesion
size, decreased growth of regenerating fibers, and increased death
of neurons spared by the initial lesion (Fitch at al., 1999;
McPhail et al. 2004; Donnelly and Popovich, 2007). The negative
effects of activated macrophages and neutrophils are thought to be
mediated by the secretion of cytokines, eicosanoids, free radicals,
and proteases, which can be toxic to both neurons and glia
(Donnelly and Popovich, 2007). Numerous studies in which
macrophages have been depleted, inhibited, or inactivated after
spinal cord injury have reported neuroprotection, increased
regeneration, and improvements in motor, sensory, and autonomic
function (Oudega at al., 1999; Popovich at al., 1999; McPhail et
al., 2004; Stirling et al., 2004).
SUMMARY OF THE INVENTION
[0008] The invention is based in part on the inventors' observation
that, in an in vitro glial scar model, axonal retraction (dieback)
ED-1.sup.+ cells, such as activated macrophages and microglia, can
he reduced by the external administration of certain types of cell
or conditioned cell culture medium in which the cells were grown.
These in vitro results were also confirmed by cells applied in an
in vivo spinal cord injury model.
[0009] The inventors observed that ED-1.sup.+ cells, such as
activated macrophages and microglia, adhered to dystrophic axons
and that this was necessary for retraction. They found that
application of the cells, or conditioned medium from the cells, to
the dystrophic axons reduced or prevented adhesion. They further
found that application of conditioned medium from culturing the
cells had neurostimulatory effects and significantly increased
neurite outgrowth/regeneration.
[0010] Accordingly, the invention is generally directed to a method
for treating (ameliorating or preventing) neuronal injury that is
associated with axonal retraction.
[0011] The invention is generally directed to a method for treating
(ameliorating or preventing) neuronal injury by promoting axonal
growth/regeneration in or around a lesion.
[0012] The invention is also generally directed to a method for
reducing axonal retraction in neuronal injury.
[0013] The invention is also generally directed to a method for
promoting axonal growth/regeneration in or around a lesion.
[0014] Retraction can be caused by ED-1.sup.+ cells, such as
activated macrophages and/or microglia
[0015] The invention is also generally directed to a method for
reducing adhesion of ED-1.sup.+ cells to dystrophic axons so as to
reduce axonal retraction.
[0016] These results are achieved by administering cells in
sufficient proximity to the lesion, for a time sufficient, and in
sufficient amount to promote axonal growth/regeneration in or
around the lesion.
[0017] These results are achieved by administering cells in
sufficient proximity to the lesion, for a time sufficient, and in
sufficient amount to reduce axonal retraction in neuronal injury
and reduce neuronal injury that is associated with axonal
retraction.
[0018] These results are achieved by administering cells in
sufficient proximity to the lesion, for time sufficient, and in
sufficient amount to reduce the adhesion of ED-1.sup.+ cells to
dystrophic axons, which adhesion would result in axonal
retraction.
[0019] The cells are introduced to injured axons so that the cells
reduce adhesion of resident ED-1.sup.+ cells to the axons.
[0020] ED-1.sup.+ cells include, but are not limited to,
macrophages and microglia.
[0021] These results are caused by factors secreted by the cells.
Therefore, the results are also achieved by using a cell
culture-conditioned medium or fractions thereof or proteins or
other factors derived from the conditioned medium. The conditioned
medium is produced by growing the cells, that are effective to
reduce adhesion and axonal retraction and/or promote axonal growth,
in cell culture. In one embodiment, the conditioned medium is not
frozen before use.
[0022] These results are also achieved using a cell lysate or
cellular fractions.
[0023] The cells, secreted factors, fractions, etc., disclosed
above, may be administered at various timepoints that correspond to
axonal retraction and the injury that results from it, such as at
the time of an acute injury, to extended periods (e.g., weeks)
after the initial acute injury.
[0024] It is understood, however, that axonal retraction may also
occur in chronic injury conditions, such as those described below
in chronic injury, the cells may be administered according to any
regimen that would reduce axonal retraction.
[0025] Because the cells (and secreted factors) also promote axonal
growth, they also may be administered in injuries, chronic and
acute, that are not necessarily associated with retraction. Such
injuries are treated so as to provide and promote axonal
growth/regeneration in or around the lesion.
[0026] In one embodiment, the cells are stem cells. Stem cells
include, but are not limited to, embryonic stem cells and
non-embryonic stem cells. The non-embryonic stem cells, like
embryonic stem cells, may have the ability to differentiate into
cell types of more than one embryonic germ layer and/or express one
or more markers associated with the potential to differentiate into
cell types of more than one embryonic germ layer. Non-embryonic
cells also include tissue-specific stem cells, i.e., that have the
ability to differentiate into cells types of only one embryonic
germ layer, for example, hematopoietic stem cells, neural stem
cells, and mesenchymal stem cells.
[0027] In a specific embodiment, the. non-embryonic stem cells have
been designated "multipotent adult progenitor cells" ("MAPC") and
are described in U.S. Pat. No. 7,015,037,
[0028] The invention encompasses any nervous system injury that
produces axonal dystrophy where ED-1.sup.+ cells, such as activated
macrophages or microglia, interact with the dystrophic axons and
cause the axons to retract. This includes tissues of the central
nervous system, including brain and spinal cord. Conditions
associated with dystrophic axons include, but are not limited to,
spinal cord injury produced by any type of traumatic influence to
the spinal cord (these include any force coming from outside the
spinal cord (including disc herniation)) or coming from within the
spinal cord, such as syringomyelia; brain injury (i.e., head
trauma) produced by any type of traumatic influence from within or
outside the brain; stroke (ischemic or hemolytic) throughout the
central nervous system; multiple sclerosis; epilepsy;
neurodegenerative diseases, such as Alzheimer's Disease,
Parkinson's Disease, amylotropic lateral sclerosis (Lou Gehrig's
Disease), and Creutzfeldt-Jakob Disease (CJD).
BRIEF DESCRIPTION OF THE FIGURES
[0029] FIG. 1--Schematic representation of regeneration failure
after spinal cord injury.
[0030] FIG. 2--Schematic representation of axonal dieback in
vivo.
[0031] FIGS. 3A-C--Actual and graphical representation of
macrophage infiltration and axonal (tieback following dorsal column
crush. Macrophage infiltration correlates with mortal retraction
after spinal cord injury. There is extensive retraction of
ascending sensory axons over time after spinal cord injury. FIGS.
3A-B are image montages of 20 .mu.m thick longitudinal sections of
a dorsal column crush (DCC) spinal cord injury 2 d (FIG. 3A) and 7
d (FIG. 3B) after lesion. Dex-TR, Texas Red conjugated dextran 3000
WV. The orientation of the sections is such that caudal is on the
left side of the image and rostral is on the right. The white boxes
below represent axonal position with respect to the lesion center
(dotted lines) with superimposed fiber tracings of multiple
sections from one animal at each time point. The ruler tick marks
indicate 200 .mu.m increments. At 2 d after lesion, dorsal root
ganglion axons (red) have retracted a short distance from the
initial site of axotomy at the lesion center, marked by GFAP+
reactive astrocytes (blue) (FIG. 3A). There are a few ED-1+ cells
(green) within the lesion, which are most likely activated
microglia. By 7 d after lesion, injured axons (red) have retracted
extensively from the lesion center (FIG. 3B). The lesion and
surrounding tissue are now filled with ED-1+ cells (green), which
are predominantly infiltrating macrophages, whereas reactive
astrocytes (blue) have vacated the lesion core. FIG. 3C is a graph
indicating average axonal retraction over time. The majority of
retraction occurred during the first 7 d after lesion; however,
retraction did continue up to 28 d after lesion, the length of time
studied. Axonal retraction (black graph) is as follows: day 2 is
significantly different from days 7, 14, 28 (one-way ANOVA,
F(4,40)=6.50, p<0.001; Tukey's post hoc test, #p<0.05,
*p<0.01, **p<0.001). Macrophage depletion (red graph) is as
follows: day 2 is significant from days 7, 14, and 28; day 4 is
significant from 14 and 28; day 7 is significant from days 2 and 14
(one-way ANOVA, F(4,40)=22.83, p<0.001; Tukey's post hoc test,
*p<0.01, **p<0.001, ***p<0.0001). Error bars indicate SEM.
Scale bars: 250 .mu.m (FIGS. 3A-B).
[0032] FIGS. 4A-C--Time-lapse montage of macrophages inducing
neuronal dieback in vitro. Macrophages induce extensive retraction
of dystrophic adult dorsal root ganglion axons in an in vitro model
of the glial scar. FIG. 4A is a six-panel montage of single-frame
images from a time-lapse movie in which NR8383 macrophages were
added to a culture of dystrophic adult dorsal root ganglion neurons
growing on an inverse spot gradient of the growth-promoting
extracellular matrix molecule laminin and the potently inhibitory
chondroitin sulfate proteoglycan aggrecan. Times for each frame are
given in the bottom right of each image, and an arrow marks the
central domain of the growth cone. An asterisk marks a consistent
point in the culture as a reference for growth cone position during
frame shifts, initial macrophage-growth cone contact was made
immediately of macrophage addition at 30 min. Physical contacts are
observed between a second macrophage and the dystrophic axon at 61
min. Additional macrophages physically altered the axonal
trajectory as retraction began at 110 min. The growth cone is
obscured by multiple macrophages and has retracted nearly out of
the frame at 150 min. Scale bar, 20 .mu.m. FIG. 4B is a positional
graph tracking the growth, cone for entire time-lapse movie in FIG.
4A. Each point represents the position of the central domain of the
growth cone for a single frame (every 30 s). The axon underwent
extensive retraction of .about.100 .mu.m after macrophage contact.
FIG. 4C is a positional graph from another representative
time-lapse experiment.
[0033] FIGS. 5A-D--Contacts formed between axons and macrophages.
Macrophages physically interact with dystrophic axons in an in
vitro model of the glial scar. FIG. 5A shows select frames from a
time-lapse movie in which macrophages physically contact a
dystrophic axon. Before retraction occurred, the growth cone was
still attached while the axon was lifted from the substrate and
severely bent (arrows). FIG. 5B is a higher magnification image of
the third image from FIG. 3A. Several adhesive contacts were made
between a macrophage and a dystrophic axon. The arrows indicate
membrane processes that formed from these contacts as the
macrophage moved away from the axon. FIG. 5C is a 40.times.
confocal z-stack three-dimensional reconstruction of a culture of
adult DRG neurons (red) 2.5 h after macrophage (green) addition. A
macrophage is observed in direct contact with the dystrophic growth
cone. FIG. 5D is a 90.degree. rotation of FIG. 5C about the x-axis
yielding a side view of the three-dimensional reconstruction. The
arrow indicates a neuronal process (red) that has been lifted from
the substrate by the adjacent macrophage (green). Scale bars: 20
.mu.m (FIGS. 5A-B); 50 .mu.m (FIG. 5C).
[0034] FIGS. 6A-C--Time-lapse montage of MMP9 inhibitor preventing
axonal dieback from macrophage contact (FIG. 6A). FIG. 6B is a
positional graph tracking the growth core for entire time-lapse
movie in FIG. 6A. FIG. 6C is a positional graph from another
representative time-lapse experiment.
[0035] FIG. 7--Experimental design to assess the effect of
externally-added living cells (MAPCs) or conditioned medium on
macrophage-induced dorsal root ganglion (DRG) neuron dieback.
[0036] FIGS. 8A-B--FIG. 8A is a time-lapse montage of MAPCs
co-cultured with DRGs showing that the addition of MAPCs prevent
macrophage-induced dieback. MAPCs are administered one day before
the addition of macrophages. FIG. 8B is a positional graph tracking
the growth cone for entire time-lapse movie in FIG. 8A.
[0037] FIGS. 9A-B--FIG. 9A is a time-lapse montage of experiment
showing that MAPC conditioned medium prevents macrophage-induced
axonal dieback. Conditioned medium is added thirty minutes prior to
the addition of macrophages. FIG. 9B is a positional graph tracking
the growth cone for entire time-lapse movie in FIG. 9A.
[0038] FIGS. 10A-B--FIG. 10A is a time-lapse montage showing that
macrophages stimulated with MAPC-conditioned medium do not induce
axonal dieback. FIG. 10B is a positional graph tracking the growth
cone for entire tune-lapse movie in FIG. 10A.
[0039] FIG. 11--Graphical representation of MAPCs preventing
macrophage-mediated axonal dieback.
[0040] FIG. 12--Experimental summary of in vitro experiments with
MAPC or conditioned medium
[0041] FIGS. 13A-B--MAPCs prevent macrophage-mediated axonal
dieback after dorsal column crush injury and promote regeneration
into the lesion core. Graphical (FIG. 13A) and actual (FIG. 13B)
representation of seven day post-injury spinal cord sections in
which a vehicle control or MAPCs were transplanted. Macrophages
induce extensive retraction of dystrophic adult dorsal root
ganglion axons in an in vitro model of the glial scar. NR838
macrophages were added to a culture of dystrophic adult dorsal root
ganglion neurons growing on an inverse spot gradient of the
growth-promoting extracellular matrix molecule laminin and the
potently inhibitory chondroitin sulfate proteoglycan aggrecan. A
positional graph tracks the growth cone for entire time-lapse
movie. Each point represents the position of the central domain of
the growth cone for a single frame (every 30 s). The axons
underwent extensive retraction of .about.100 .mu.m after macrophage
contact.
[0042] The panels in FIG. 13B show a 10.times. image montages of 20
.mu.m thick longitudinal sections of a dorsal column crush (DCC)
spinal cord injury 7 d after lesion. Fibers are labeled with Texas
Red-conjugated 3000 MW dextran and macrophages are visualized with
ED-1+ (purple). The orientation of the sections is such that caudal
is on the left side of the image and rostral is on the right. The
lesion center is marked below (solid black lines) with three
superimposed fiber tracings of multiple sections from one animal
for each condition. At 7 days after lesion and vehicle injection
only, dorsal root ganglion axons (red) have retracted extensively
distance from the initial site of axotomy at the lesion center.
(FIG. 13A). By 7 d after lesion and MAPC transplant, injured axons
have regenerated into the lesion in large numbers (13B). FIG. 13A
is a graph indicating average axonal retraction over 2, 4, and 7
days after injury in animals receiving vehicle control or MAPC
transplants. The conditions, MAPC transplant versus Vehicle
control, are significantly different from each other by General
Linear Model, *p<0.0001. Scale Bar: 200 .mu.m (FIGS. 13A-B).
[0043] FIGS. 14A-C--FIG. 14A is a time-lapse montage showing that
NG2.sup.+ glial cells do not prevent macrophage-induced axonal
retraction. NG2+ cells stabilize axons, but do not prevent
macrophage-mediated retraction following macrophage attack in
vitro. FIG. 14A is six representative frames from a time-lapse
movie illustrating macrophage/axon interactions on an
aggrecan/laminin gradient in the presence of adult mouse spinal
cord NG2.sup.+ cells. NR8383 macrophages are added to a 2 DIV
culture of adult DRG neurons. Times for each frame are given in the
lower right of each image and an asterisk marks a consistent point
on the culture dish as a reference for position during frame
shifts. An arrow denotes the central domain of the grown cone.
Macrophages are added following a 30 minute period of observation
and first contact occurs at 103'. The axon has already undergone a
long distance retraction by 110'. Open arrow indicates the presence
of a retraction fiber. FIG. 14B is a graph of growth cone position
for each frame (30 sec) of the time lapse movie shown in FIG. 14A.
Red are represents the location of the inner rim of the spot. Arrow
indicates initial trajectory of growth. FIG. 14C shows the distance
from the origin of six dystrophic axons in co-culture with NG2+
cells on the aggrecan/laminin spot gradient following contact with
macrophages. An arrowhead indicates the position at which the axon
has retracted to an NG2 cell. Scale Bar: 20 .mu.m (FIG. 14A).
[0044] FIGS. 15A-B Confocal image of MAPCs cultured on a spot
gradient alone and higher magnification image of MAPCs growing with
neurons on the spot gradient.
[0045] FIG. 15A is a 10.times. confocal image of GFP+ MAPC (green)
cultured on a bidirectional gradient of aggrecan, visualized by
CS56 (red), and laminin. FIG. 15B is a 40.times. confocal image of
MAPC co-cultured with adult DRG neurons visualized by
.beta.-tubulin (blue). Both adult DRGs and MAPCs do not cross the
inhibitory spot rim after 2 days in vitro.
[0046] MAPCs added to the aggrecan spot gradient did not invade the
inhibitory rim, but adhered well the center of the spot and
associated with adult DRG axons.
[0047] FIGS. 16A-C--Graphical (FIG. 16A) and actual (FIGS. 16B-C)
representation of the effect of control media or MAPC-conditioned
media on axon outgrowth in vitro.
[0048] FIGS. 17A-B--FIG. 17A is a time-lapse montage of experiment
showing that control medium does not prevent macrophage-induced
axonal dieback. Conditioned medium is added thirty minutes prior to
the addition of macrophages.
[0049] Macrophages induce extensive retraction of dystrophic adult
dorsal root ganglion axons in an in vitro model of the glial scar
despite the presence of control MAPC media. FIG. 17A is a six-panel
montage of single-frame images from a time-lapse movie in which
NR8383 macrophages were added to a culture of dystrophic adult
dorsal root ganglion neurons growing on an inverse spot gradient of
the growth-promoting extracellular matrix molecule laminin and the
potently inhibitory chondroitin sulfate proteoglycan aggrecan.
Times for each frame are given in the bottom right of each image,
and an arrow marks the central domain of the growth cone. An
asterisk marks a consistent point in the culture as a reference for
growth cone position during frame shifts. Scale bar, 20 .mu.m. FIG.
17B is a positional graph tracking the growth cone for entire
time-lapse movie in A. Each point represents the position of the
central domain of the growth cone for a single frame (every 30 s).
The axon underwent extensive retraction of .about.80 .mu.m after
macrophage contact.
[0050] Direct addition of MAPC-conditioned media to the timelapse
dish resulted in a change in growth cone morphology, from a
dystrophic, stalled state, to as motile, flattened state.
Macrophages still contacted these axons, but contorts were
generally transient and generally did not result in axonal
retraction. Control MAPC media did not prevent axonal retraction.
Macrophages pretreated with MAPC-conditioned media also contacted
axons on the spot, but did not cause retraction (FIGS. 9A-12). It
is possible that MAPCs act on macrophages to alter their receptor
expression, response to injured cells, or secretion of MMP-9.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0051] "A" or "an" means herein one or more than one; at least one.
Where the plural form is used herein, it generally includes the
singular.
[0052] As used herein, the terms "adhere(s), adherence, adhesion",
and the like, refer to an association of sufficient duration so as
to induce axonal retraction. As described further herein, it is
understood that physical contact may occur between macrophages (or
other cells) and dystrophic axons that is transient and does not
result in axonal retraction. Within the context of the invention,
the adherence that is reduced or prevented by the reagents of the
invention is that which occurs for sufficient duration so as to
induce axonal retraction. Thus, the invention does not exclude
reagents that allow physical contact between dystrophic axons and
ED-1.sup.+ cells. The invention thus encompasses reagents that
allow contact (such as transient physical contact) but do not allow
adherence for time sufficient to result in axonal dieback.
[0053] "Co-administer" means to administer in conjunction with one
another, together, coordinately, including simultaneous or
sequential administration of two or more agents.
[0054] "Comprising" means, without other limitation, including the
referent, necessarily, without any qualification or exclusion on
what else may be included. For example, "a composition comprising x
and y" encompasses any composition that contains x and y, no matter
what other components may be present in the composition. Likewise,
"a method comprising the step of x" encompasses any method in which
x is carried out, whether x is the only step in the method or it is
only one of the steps, no matter how many other steps there may be
and no matter how simple or complex x is in comparison to them.
"Comprised of and similar phrases using words of the root
"comprise" are used herein as synonyms of "comprising" and have the
same meaning.
[0055] "Comprised of" is a synonym of comprising (see above).
[0056] "Conditioned cell culture medium" is a term well-known the
art and refers to medium in which cells have been grown. Herein
this means that the cells are grown for a sufficient time to
secrete the factors that are effective to reduce the adhesion of
activated macrophages to dystrophic neurons and/or promote neurite
outgrowth/axon regeneration.
[0057] Conditioned cell culture medium refers to medium in which
cells have been cultured so as to secrete factors into the medium.
For the purposes of the present invention, cells can he grown
through a sufficient number of cell divisions so as to produce
effective amounts of such factors so that the medium reduces the
adhesion of macrophages to dystrophic neurons and hence reduces
axonal retraction and/or promote neurite outgrowth/axon
regeneration. Cells are removed from the medium by any of the known
methods in the art, including, but not limited to, centrifugation,
filtration, immunodepletion (e.g., via tagged antibodies and
magnetic columns), and FACS sorting.
[0058] "Dieback" is a term of art used to refer to axonal
retraction that occurs as a result of trauma to the axon. The
axonal retraction, within the context of the invention, refers to
that which occurs as a result of sufficient adherence of ED-1.sup.+
cells and, particularly, macrophages and microglia. Such
macrophages and microglia (i.e., ED-1.sup.+ cells) are not in the
resting or inactive state. They are activated. The term "activated"
refers to a state of these cells that allows them to adhere to a
dystrophic axon so as to result in axonal retraction. Examples of
conditions resulting in activation in vitro are described further
in this application. It is to be understood, however, that such
activation is not limited to the specific conditions disclosed
herein.
[0059] Although the invention is often specifically directed to
(and exemplified by) activated macrophages, these are a class of
ED-1.sup.+ cells and the invention pertains to other such cells.
One example is activated microglia.
[0060] "Effective amount" generally means an amount which provides
the desired local or systemic effect. For example, an effective
amount is an amount sufficient to effectuate a beneficial or
desired clinical result. The effective amounts can be provided all
at once in a single administration or in fractional amounts that
provide the effective amount in several administrations. The
precise determination of what would be considered an effective
amount may be based on factors individual to each subject,
including their size, age, injury, and/or disease or injury being
treated, and amount of time since the injury occurred or the
disease began. One skilled in the art will be able to determine the
effective amount for a given subject based on these considerations
which are routine in the art. As used herein, "effective dose"
means the seine as "effective amount."
[0061] "Effective route" generally means a route which provides for
delivery of an agent to a desired compartment, system, or location.
For example, an effective route is one through which an agent can
be administered to provide at the desired site of action an amount
of the agent sufficient to effectuate ,a beneficial or desired
clinical result.
[0062] "EC cells" were discovered from analysis of a type of cancer
called a teratocarcinoma. In 1964, researchers noted that a single
cell in teratocarcinomas could be isolated and remain
undifferentiated in culture. This type of stem cell became known as
an embryonic carcinoma cell (EC cell).
[0063] "Embryonic Stern Cells (ESC)" are well known in the art and
have been prepared from many different mammalian species for many
years. Embryonic stem cells are stein cells derived from the inner
cell mass of an early stage embryo known as a blastocyst. They are
able to differentiate into all derivatives of the three primary
germ layers: ectoderm, endoderm, and mesoderm. These include each
of the more than 220 cell types in the adult body. The ES cells can
become any tissue in the body, excluding placenta. Only the
morula's cells are totipotent, able to become all tissues and a
placenta.
[0064] Use of the term "includes" is not intended to be limiting.
For example, stating that the antibody inhibitor "includes"
fragments and variants does not mean that other forms of the
antibody inhibitor are excluded.
[0065] "Induced pluripotent stem cells (IPSC or IPS cells)" are
somatic cells that have been reprogrammed, for example, by
introducing exogenous genes that confer on the somatic cell a less
differentiated phenotype. These cells can then he induced to
differentiate into less differentiated. progeny. IPS cells have
been derived using modifications of an approach originally
discovered in 2006 (Yamanaka, S. at al., Cell Stem Cell, 1:3949
(2007)). For example, in one instance, to create IPS cells,
scientists started with skin cells that were then modified by a
standard laboratory technique using retroviruses to insert genes
into the cellular DNA. In one instance, the inserted genes were
Oct4, Sox2, Lif4, and c-myc, known to act together as natural
regulators to keep cells in an embryonic stem cell-like state.
These cells have been described in the literature. See, for
example, Wernig at al., PNAS, 105;5856-5861 (2008); Jaenisch at
al., Cell, 132:567-582 (2008); Hanna at al., Cell, 133:250-264
(2008); and Brambrink et al., Cell Stem Cell, 2:151459 (2008).
These references are incorporated by reference for teaching IPSCs
and methods for producing them. It is also possible that such cells
can be created by specific culture conditions (exposure to specific
agents).
[0066] The term "isolated" refers to a cell or cells which are not
associated with one or more cells or one or more cellular
components that are associated with the cell or cells in viva. An
"enriched population" means a relative increase in numbers of a
desired cell relative to one or more other cell types in viva or in
primary culture.
[0067] However, as used herein, the term "isolated" does not
indicate the presence of only stem cells. Rather, the term
"isolated" indicates that the cells are removed from their natural
tissue environment and are present at a higher concentration as
compared to the normal tissue environment. Accordingly, an
"isolated" cell population may further include cell types in
addition to stem cells and may include additional tissue
components. This also can be expressed in terms of cell doublings,
for example. A cell may have undergone 10, 20, 30, 40 or more
doubling in vitro or ex vivo so that it is enriched compared to its
original numbers in vivo or in its original tissue environment
(e.g., bone marrow, peripheral blood, adipose tissue, etc.).
[0068] "MAPC" is an acronym for "multipotent adult progenitor
cell." It refers to a non-embryonic stem cell. The term "adult" in
MAPC is non-restrictive. It refers to a non-embryonic somatic cell.
Like embryonic stem cells, the MAPC can give rise to cell lineages
of more than one germ layer. It may give rise to cell types of all
three germ layers (i.e., endoderm, mesoderm and ectoderm) upon
differentiation. Like embryonic stem cells, human MAPCs express
telomerase, Oct 3/4 (i.e., Oct 3A), rex-1, rox-1 and sox-2, and may
express SSEA-4, (See also Jiang, Y. at Nature, 418:41 (2002); Exp
Hematol 30:896 (2002)). The telomeres are extended in MAPCs and
they are karyotypically normal. Because MAPCs injected into a
mammal can migrate to and assimilate within multiple organs, MAPCs
are self-renewing stem cells. "Multipotent", with respect to MAPC,
refers to the ability to give rise to cell lineages of more than
more than one primitive germ layer (i.e., endoderm, mesoderm and
ectoderm) upon differentiation, such as all three.
[0069] "Neurite outgrowth" refers to the property of neurons at the
site of the injury not only to cease to retract but to grow and
extend.
[0070] "Pharmaceutically acceptable carrier" is any
pharmaceutically acceptable medium for the cells used in the
present invention. Such a medium may retain isotonicity, cell
metabolism, pH, and the like. It is compatible with administration
to a subject in vivo, and can be used, therefore, for cell delivery
and treatment.
[0071] "Primordial embryonic germ cells" (PG or EG cells) can be
cultured and stimulated to produce many less differentiated cell
types.
[0072] "Progenitor cells" are cells produced during differentiation
of a stem cell that have some, but not all, of the characteristics
of their terminally-differentiated progeny. Defined progenitor
cells, such as "cardiac progenitor cells," are committed to a
lineage, but not to a specific or terminally differentiated cell
type. The term "progenitor" as used in the acronym "MAPC" does not
limit these cells to a particular lineage.
[0073] The term "reduce" as used herein means to prevent as well as
decrease, in the context of treatment, to "reduce" is to both
prevent or ameliorate one or more clinical symptoms. A clinical
symptom is one (or more) that has or will have, if left untreated,
a negative impact on the quality of life (health) of the
subject.
[0074] The terra "retraction" refers to the receding of the axon
away from the site of injury, such as from where the glial scar
forms. Here, the end of regenerating axons stop extending and
become dystrophic. These dystrophic ends then can recede further
from the glial scar and the site of injury.
[0075] "Self-renewal" refers to the ability to produce replicate
daughter stem cells having differentiation potential that is
identical to those front which they arose. A similar term used in
this context is "proliferation."
[0076] "Stem cell" means a cell that can undergo self-renewal
(i.e., progeny with the same differentiation potential) and also
produce progeny cells that are more restricted in differentiation
potential. Within the context of the invention, a stem cell would
also encompass a more differentiated cell that has
dedifferentiated, for example, by nuclear transfer, by fusions with
a more primitive stem cell, by introduction of specific
transcription factors, or by culture under specific conditions.
See, for example, Wilmut et al., Nature, 385:810-813 (1997); Ying
et al., Nature, 416:545-548 (2002); Guan et al., Nature,
440;1199-1203 (2006) Takahashi et al., Cell, 126:663-676 (2006);
Okita et al., Nature, 448:313-317 (2007); and Takahashi et al.,
Cell, 131;861-872 (2007).
[0077] Dedifferentiation may also be caused by the administration
of certain compounds or exposure to a physical environment in vitro
or in vivo that would cause the dedifferentiation. Stem cells also
may be derived from abnormal tissue, such as a teratocarcinoma and
some other sources such as embryoid bodies (although these can be
considered embryonic stem cells in that they are derived from
embryonic tissue, although not directly from the inner cell mass).
Stem cells may also be produced by introducing genes associated
with stem cell function into a non-stem cell, such as an induced
pluripotent stem cell.
[0078] "Subject" means a vertebrate, such as a mammal, such as a
human. Mammals include, but are not limited to, humans, dogs, cats,
horses, cows, and pigs.
[0079] The term "therapeutically effective amount" refers to the
amount determined to produce any therapeutic response in a mammal.
For example, effective amounts of the therapeutic cells or
cell-associated agents may prolong the survivability of the
patient, and/or inhibit overt clinical symptoms. Treatments that
are therapeutically effective within the meaning of the term as
used herein, include treatments that improve a subject's quality of
life even if they do not improve the disease outcome per se. Such
therapeutically effective amounts are readily ascertained by one of
ordinary skill in the art. Thus, to "treat" means to deliver such
an amount. Thus, treating can prevent or ameliorate any
pathological symptoms that occur from the adherence of activated
macrophages to dystrophic axons. Treating also refers to the
beneficial clinical effect of axon regeneration.
[0080] "Treat," "treating," or "treatment" are used broadly in
relation to the invention and each such term encompasses, among
others, preventing, ameliorating, inhibiting, or curing a
deficiency, dysfunction, disease, or other deleterious process,
including those that interfere with and/or result from a
therapy.
Stem Cells
[0081] The present invention can he practiced, preferably, using
stern cells of vertebrate species, such as humans, non-human
primates, domestic animals, livestock, and other non-human mammals.
These include, but are not limited to, those cells described
below.
[0082] Embryonic Stem Cells
[0083] The most well studied stein cell is the embryonic stem cell
(ESC) as it has unlimited self-renewal and multipotent
differentiation potential. These cells are derived from the inner
cell mass of the blastocyst or can be derived from the primordial
germ cells of a post-implantation embryo (embryonal germ cells or
EG cells). ES and EG cells have been derived, first from mouse, and
later, from many different animals, and more recently, also from
non-human primates and humans. When introduced into mouse
blastocysts or blastocysts of other animals, ESCs can contribute to
all tissues of the animal. ES and EG cells can be identified by
positive staining with antibodies against SSEA1 (mouse) and SSEA4
(human). See, for example, U.S. Pat. Nos. 5,453,357; 5,656,479;
5,670,372; 5,543,780; 5,874,301; 5,914,268; 6,110,739 6,190,910;
6,200,806; 6,432,711; 6436,701, 6,500,668; 6,703,1'79; 6,875,607;
7,029,913; 7,112,437; 7,145,057; 7,153,684; and 7,294,508, each of
which is incorporated by reference for teaching embryonic stem
cells and methods of making and expanding them. Accordingly, ESCs
and methods for isolating and expanding them are well-known in the
art.
[0084] A number of transcription factors and exogenous cytokines
have been identified that influence the potency status of embryonic
stem cells in vivo, The first transcription factor to be described
that is involved in stern cell pluripotency is Oct4, Oct4 belongs
to the POU (Pit-Oct-Unc) family of transcription factors and is a
DNA binding protein that is able to activate the transcription of
genes, containing an octameric sequence called "the octamer motif"
within the promoter or enhancer region, Oct4 is expressed at the
moment of the cleavage stage of the fertilized zygote until the egg
cylinder is formed. The function of Oct3/4 is to repress
differentiation inducing genes (i.e., FoxaD3, hCG) and to activate
genes promoting pluripotency (FGF4, Utf1, Rex1). Sox2, a member of
the high mobility group (HMG) box transcription factors, cooperates
with Oct4 to activate transcription of genes expressed in the inner
cell mass. It is essential that Oct3/4 expression in embryonic stem
cells is maintained between certain levels. Overexpression or
downregulation of >50% of Oct4 expression level will alter
embryonic stem cell fate, with the formation of primitive
endoderm/mesoderm or trophectoderm, respectively. In vivo, Oct4
deficient embryos develop to the blastocyst stage, but the inner
cell mass cells are not pluripotent. Instead they differentiate
along the extraembryonic trophoblast lineage. Sal14, a mammalian
Spalt transcription factor, is an upstream regulator of Oct4, and
is therefore important to maintain appropriate levels of Oct4
during early phases of embryology. When Sal14 levels fall below a
certain threshold, trophectodermal cells will expand ectopically
into the inner cell mass. Another transcription factor required for
pluripotency is Nanog, named after a celtic tribe "Tir Nan Og": the
land of the ever young. In vivo, Nanog is expressed from the stage
of the compacted morula, is subsequently defined to the inner cell
mass and is downreplated by the implantation stage. Downregulation
of Nanog may be important to avoid an uncontrolled expansion of
pluripotent cells and to allow multilineage differentiation during
gastrulation. Nanog null embryos, isolated at day 5.5, consist of a
disorganized blastocyst, mainly containing extraembryonic endoderm
and no discernable epiblast.
[0085] Non-Embryonic Stem Cells
[0086] Stem cells have been identified in most tissues. Perhaps the
best characterized is the hematopoietic stem cell (HSC). HSCs are
mesoderm-derived cells that can be purified using cell surface
markers and functional characteristics. They have been isolated
from bone marrow, peripheral blood, cord blood, fetal liver, and
yolk sac. They initiate hematopoiesis and generate multiple
hematopoietic lineages. When transplanted into lethally-irradiated
animals, they can repopulate the erythroid neutrophil-macrophage,
megakaryocyte, and lymphoid hematopoietic cell pool. They can also
be induced to undergo some self-renewal cell division. See, for
example, U.S. Pat. Nos. 5,635,387; 5,460,964; 5,677,136; 5,750,397;
5,681,599; and 5,716,827, U.S. Pat. No. 5,192,553 reports methods
for isolating human neonatal or fetal hematopoietic, stem or
progenitor cells. U.S. Pat. No. 5,716,827 reports human
hematopoietic cells that are Thy-1.sup.+ progenitors, and
appropriate growth media to regenerate them in vitro. U.S. Pat. No.
5,635,387 reports a method and device for culturing human
hematopoietic cells and their precursors. U.S. Pat. No. 6,015,554
describes a method of reconstituting human lymphoid and dendritic
cells, Accordingly, HSCs and methods for isolating and expanding
them are well-known in the art.
[0087] Another stem cell that is well-known in the art is the
neural stem cell (NSC). These cells can proliferate in vivo and
continuously regenerate at least some neuronal cells. When cultured
ex vivo, neural stem cells can he induced to proliferate as well as
differentiate into different types of neurons and glial cells, When
transplanted into the brain, neural stem cells can engraft and
generate neural and glial cells. See, for example, Gage F. H.,
Science, 287:1433-1438 (2000), Svendsen S. N. et al. Brain
Pathology, 9:499-513 (1999), and Okabe S. et al., Mech Development,
5989-102 (1996). U.S. Pat. No. 5,851,832 reports multipotent neural
stem cells obtained from brain tissue, U.S. Pat. No. 5,766,948
reports producing neuroblasts from newborn cerebral hemispheres.
U.S. Pat. Nos. 5,564,183 and 5,849,553 report the use of mammalian
neural crest stem cells, U.S. Pat. No. 6,040,180 reports in vitro
generation of differentiated neurons from cultures of mammalian
multipotential CNS stem cells, WO 98/50526 and WO 99/01159 report
generation and isolation of neuroepithelial stem cells,
oligodendrocyte-astrocyte precursors, and lineage-restricted
neuronal precursors. U.S. Pat. No. 5,968,829 reports neural stem
cells obtained from embryonic forebrain. Accordingly, neural stem
cells and methods for making and expanding them are well-known in
the art.
[0088] Another stem cell that has been studied extensively in the
art is the mesenchymal stem cell (MSC). MSCs are derived from the
embryonal mesoderm and can be isolated from many sources, including
adult bone marrow, peripheral blood, fat, placenta, and umbilical
blood, among others. MSCs can differentiate into many mesodermal
tissues, including muscle, bone, cartilage, fat, and tendon. There
is considerable literature on these cells. See, for example, U.S.
Pat. Nos. 5,486,389; 5,827,735; 5,811,094; 5,736,396; 5,837,539;
5,837,670; and 5,827,740. See also Pittenger, M. at al, Science,
284:143-147 (1999).
[0089] Another example of an adult stem cell is adipose-derived
adult stem cells (ADSCs) which have been isolated from fat,
typically by liposuction followed by release of the ADSCs using
collagenase. ADSCs are similar in many ways to MSCs derived from
bone marrow, except that it is possible to isolate many more cells
from fat. These cells have been reported to differentiate into
bone, fat, muscle, cartilage, and neurons. A method of isolation
has been described in U.S. 2005/0153442,
[0090] Other stem cells that are known in the art include
gastrointestinal stem cells, epidermal stem cells, and hepatic stem
cells, which have also been termed "oval cells" (Patten, C., at
al., Trans B Soc Lond B Bioi Sci, 353:821-830 (1998), Watt, F.,
Trans R Sac Lond B Biol Sci, 353:831 (1997); Alison at al.,
Hepatology, 29:678-683 (1998).
[0091] Other non-embryonic cells reported to be capable of
differentiating into cell types of more than one embryonic germ
layer include, but are not limited to, cells from umbilical cord
blood (see U.S. Publication No. 2002/0164794), placenta (see U.S.
Publication No. 2003/0181269, umbilical cord matrix (Mitchell, K.
E. at al., Stem Cells, 21:50-60 (2003)), small embryonic-dike stem
cells (Kucia, M. at al. J Physiol Pharmacol, 57 Suppl 5:5-18
(2006)), amniotic fluid stem cells (Atala, A., J Tissue Regan Med,
1:83-96 (2007)), skin-derived precursors (Toma et al., Nat Cell
Biol, 3:778-784 (2001)), and bone marrow (see U.S. Publication Nos.
2003/0059414 and 2006/0147246), each of which is incorporated by
reference for teaching these cells.
[0092] Strategies of Reprogramming Somatic Cells
[0093] Several different strategies such as nuclear
transplantation, cellular fusion, and culture induced reprogramming
have been employed to induce the conversion of differentiated cells
into an embryonic state. Nuclear transfer involves the injection of
a somatic nucleus into an enucleated oocyte, which, upon transfer
into a surrogate mother, can give rise to a clone ("reproductive
cloning"), or, upon explantation in culture, can give rise to
genetically matched embryonic stem (ES) cells ("somatic cell
nuclear transfer," SCNT). Cell fusion of somatic cells with ES
cells results in the generation of hybrids that show all features
of pluripotent ES cells. Explantation of somatic cells in culture
selects for immortal cell lines that may be pluripotent or
multipotent. At present, spermatogonial stem cells are the only
source of pluripotent cells that can be derived from postnatal
animals. Transduction of somatic cells with defined factors can
initiate reprogramming to a pluripotent state. These experimental
approaches have been extensively reviewed (Hoehedlinger and
Jaenisch, Nature, 441:1061-1067 (2006) and Yamanaka, S., Cell Stem
Cell, 1:39-49 (2007)).
[0094] Nuclear Transfer
[0095] Nuclear transplantation (NT), also referred to as somatic
cell nuclear transfer (SCNT), denotes the introduction of a nucleus
from a donor somatic cell into an enucleated ogocyte to generate a
cloned animal such as Dolly the sheep (Wilmut et. al., Nature,
385:810-813 (1997). The generation of live animals by NT
demonstrated that the epigenetic state of somatic cells, including
that of terminally differentiated cells, while stable, is not
irreversible fixed hat can be reprogrammed to an embryonic state
that is capable of directing development of a now organism. In
addition to providing an exciting experimental approach for
elucidating the basic epigenetic mechanisms involved in embryonic
development and disease, nuclear cloning technology is of potential
interest for patient-specific transplantation medicine.
[0096] Fusion of Somatic Cells and Embryonic Stem Cells
[0097] Epigenetic reprogramming of somatic nuclei to an
undifferentiated state has been demonstrated in murine hybrids
produced by fusion of embryonic cells with somatic cells. Hybrids
between various somatic cells and embryonic carcinoma cells
(Softer, D., Nat Rev Genet, 7:319-327 (2006), embryonic germ (EG),
or ES cells (Zwaka and Thomson, Development, 132:227-233 (2005))
share many features with the parental embryonic cells, indicating
that the pluripotent phenotype is dominant in such fusion products.
As with mouse (Tada et al., Curr Biol, 11:1553-1558 (2000)), human
ES coils have the potential to reprogram somatic nuclei after
fusion (Cowan et al. Science, 309:1369-1373(2005)); Yu et al.,
Science, 318:1917-1920 (2006)), Activation of silent pluripoteney
markers such as Oct4 or reactivation of the inactive somatic X
chromosome provided molecular evidence for reprogramming of the
somatic genome in the hybrid cells. It has been suggested that DNA
replication is essential for the activation of pluripatency
markers, which is first observed 2 days after fusion (Do and
Scholer, Stem Cells, 22:941-949 (2004)), and that forced
overexpression of Nanog in ES cells promotes pluripotency when
fused with neural stem cells (Silva et al., Nature, 441:997-1001
(2006)).
[0098] Culture-Induced Reprogramming
[0099] Pluripotent cells have been derived from embryonic sources
such as blastomeres and the inner cell mass (ICM) of the blastocyst
(ES cells), the epiblast (EpiSC cells), primordial germ cells (EG
cells), and postnatal spermatogonial stem cells ("maGSCsm"
"ES-like" cells). The following pluripotent cells, along with their
donor cell/tissue is as follows: parthogenetic ES cells are derived
from murine oocytes (Narasimha et al., Cure Biol. 7:881-884
(1997)); embryonic stem cells have been derived from blastomeres
(Wakayama et al., Stem Cells, 25:986-993 (2007)); inner cell mass
cells (source not applicable) (Eagan et al. Nature, 428:44.49
(2004)); embryonic germ and embryonal carcinoma, cells have been
derived from primordial gem cells (Matsui et al., Cell, 70;841-847
(1992)); GMCS, maSSC, and MASC have been derived from
spermatogonial stem cells (Guan et al., Nature, 440;1199-1203
(2006); Kanatsu-Shinohara et al., Cell, 119;1001-1012 (2004); and
Seandel et al., Nature, 449:346-350 (2007)); EpiSC cells are
derived from epiblasts (Brous at al., Nature, 448;191-195 (2007);
Tesar at al., Nature, 448;196-199(2007)); pathogenetic ES cells
have been derived from human oocytes (Cibelli at al., Science,
295L819 (2002); Revazova at al., Cloning Stem Cells, 9:432449
(2007)); human ES cells have bean derived from human blastocysts
(Thomson at al., Science, 282:1145-1147 (1998)); MAPC have been
derived from bone marrow (Jiang at al., Nature, 418:41-49 (2002);
Phinney and Prockop, Stem Cells, 25:2896-2902 (2007)); cord blood
cells (derived from cord blood) (van de Ven at al., Exp Hematol,
35:1753-1765 (2007)); neurosphere derived cells derived from neural
cell (Clarke at al., Science, 288:1660-1663 (2000)). Donor cells
from the germ cell lineage such as PGCs or spermatogonial stem
cells are known to be unipotent in vivo, but it has been shown that
pluripotent ES-like cells (Kanatsu-Shinohara at al., Cell,
119:1001-1012 (2004) or maGSCs (Guan at al., Nature, 440:1199-1203
(2006), can be isolated alter prolonged in vitro culture, While
most of these pluripotent cell types were capable of in vitro
differentiation and teratoma formation, only ES, EG, EC, and the
spertriatogonial stem cell-derived maGCSs or ES-like cells were
pluripotent by more stringent criteria, as they were able to form
postnatal chimeras and contribute to the germline. Recently,
multipotent adult spermatogonial stem cells (MASCs) were derived
from testicular spermatogonial stem cells of adult mice, and these
cells had an expression profile different from that of ES cells
(Seandei et al., Nature, 449:346-350 (2007)) but similar to EpiSC
cells, which were derived from the epiblast of postimplantation
mouse embryos (Brons et al., Nature, 448:191-195 (2007); Tesar et
al., Nature, 448:196-199 (2007)).
[0100] Reprogramming by Defined Transcription Factors
[0101] Takahashi and Yamanaka have reported reprogramming somatic
cells back to an ES-like state (Takahashi and Yamanaka, Cell,
126;663-676 (2006)) They successfully reprogrammed mouse embryonic
fibroblasts (MEFs) and adult fibroblasts to pluripotent cells after
viral-mediated transduction of the four transcription factors Oct4,
Sox2, c-myc, and Klf4 followed by selection for activation of the
Oct4 target gene Fbx15, Cells that had activated Fbx15 were coined
iPS (induced pluripotent stem) cells and were shown to be
pluripotent by their ability to form teratomas, although the were
unable to generate live chimeras. This pluripotent state was
dependent on the continuous viral expression of the transduced Oct4
and Sox2 genes, whereas the endogenous Oct4 and Nanog genes were
either not expressed or were expressed at a lower level than in ES
cells, and their respective promoters were found. to he largely
methylated. This is consistent with the conclusion that the
Fbx15-iPS cells did not correspond to ES cells but may have
represented an incomplete state of reprogramming. While genetic
experiments had established that Oct4 and Sox2 are essential for
pluripoteney (Chambers and Smith, Oncogene, 23:7150-7160 (2004);
Ivanona et al., Nature, 442;53305318 (2006); Masui et al., Nat Cell
Biol, 9:625-635 (2007)), the role of the two oncogenes c-myc and
Klf4 in reprogramming is less clear. Some of these oncogenes may,
in fact, be dispensable for reprogramming, as both mouse and human
iPS cells have been obtained in the absence of c-myc transduction,
although with low efficiency (Nakagawa at al., Nat Biotechnol,
26:191-106 (2008); Werning et al., Nature, 448:318-324 (2008); Yu
et al., Science, 318: 1917-1920 (2007)).
MAPC
[0102] MAPC is an acronym for "multipotent adult progenitor cell"
(non-ES, non-EG, non-germ), MAPC have the capacity to differentiate
into cell types of at least two, such as, all three, primitive germ
layers (ectoderm, mesoderm, and endoderm). Genes found in ES cells
were also fiend in MAPC telomerase, Oct 3/4, rex-1, rox-1, sox-2).
Oct 3/4 (Oct 3A in humans) appears to be specific for ES and germ
cells. MAPC represents a more primitive progenitor cell population
than MSC and demonstrates differentiation capability encompassing
the epithelial, endothelial, neural, myogenic, hematopoietic,
osteogenic, hepatogenic, chondrogenic and adipogenic lineages
(Verfaillie, C. M., Trends Cell Biol 12:502-8 (2002), Jahagirdar,
B. N., et al., Exp Hematol, 29:543-56 (2001); Reyes, M. and C. M.
Verfaillie, Ann N Y Acad Sci, 938;231-233 (2001); Jiang, Y. et al.,
Exp Hematol, 30896-904 (2002); and (Jiang, Y. at al., Nature,
418:41-9, (2002)).
[0103] Human MAPCs are described in U.S. Pat. No. 7,015,037 and
U.S. application Ser. No. 10/467,963. MAPCs have been identified in
other mammals. Murine MAPCs, for example, are also described in
U.S. Pat. 7,015,037 and U.S. application Ser. No. 10/467,963. Rat
MAPCs are also described in U.S. application Ser. No.
10/467,963.
[0104] These references are incorporated by reference for
describing MAPCs isolated by Catherine Verfaillie.
Isolation and Growth of MAPCs
[0105] Methods of MAPC isolation are known in the art. See, for
example, U.S. Pat. No. 7,015,037 and U.S. application Ser. No.
10/467,963, and these methods, along with the characterization
(phenotype) of MAPCs, are incorporated herein by reference. MAPCs
can be isolated from multiple sources, including, but not limited
to, bone marrow, placenta, umbilical cord and cord blood, muscle,
brain, liver, spinal cord, blood or skin. It is, therefore,
possible to obtain bone marrow aspirates, brain or liver biopsies,
and other organs, and isolate the cells using positive or negative
selection techniques available to those of skill in the art,
relying upon the genes that are expressed (or not expressed) in
these cells (e.g., by functional or morphological assays such as
those disclosed in the above-referenced applications, which have
been incorporated herein by reference).
[0106] MAPCs from Human Bone Marrow as Described in U.S. Pat. No.
7,015,037
[0107] MAPCs do not express the common leukocyte antigen CD45 or
erythroblast specific glycophoria-A (Gly-A). The mixed population
of cells was subjected to a Ficoll Hypaque separation. The cells
were then subjected to negative selection using anti-CD45 and
anti-Gly-A antibodies, depleting the population of CD45.sup.+ and
Gly-A.sup.+ cells, and the remaining approximately 0.1% of marrow
mononuclear cells were then recovered. Cells could also be plated
in fibronectin-coated well and cultured as described below for 2-4
weeks to deplete the cells of CD45.sup.+ and Gly-A.sup.+ cells. In
cultures of adherent bone marrow cells, many adherent stromal cells
undergo replicative senescence around cell doubling 30 and a more
homogenous population of cells continues to expand and maintains
long telomeres.
[0108] Alternatively, positive selection could be used to isolate
cells via a combination of cell-specific markers. Both positive and
negative selection techniques are available to those of skill in
the art, and numerous monoclonal and polyclonal antibodies suitable
for negative selection purposes are also available in the art (see,
for example, Leukocyte Typing V, Schlossman, et al., Eds. (1995)
Oxford University Press) and are commercially available from a
number of sources.
[0109] Techniques for mammalian cell separation from a mixture of
cell populations have also been described by Schwartz, et al., in
U.S. Pat. No. 5,759,793 (magnetic separation), Basch at al., 1983
(immunoaffinity chromatogaphy), and Wysocki and Sato, 1978
(fluorescence-activated cell sorting).
Culturing MAPCs as Described in U.S. Pat. No. 7,015,037
[0110] MAPCs isolated as described herein can be cultured using
methods disclosed herein and in U.S. Pat. No. 7,015,037, which is
incorporated by reference for these methods.
Additional Culture Methods
[0111] In additional experiments the density at which MAPCs are
cultured can vary from about 100 cells/cm.sup.2 or about 150
cells/cm.sup.2 to about 10,000 cells/cm.sup.2, including about 200
cells/cm.sup.2 to about 1500 cells/cm.sup.2to about 2000
cells/cm.sup.2. The density can vary between species. Additionally,
optimal density can vary depending on culture conditions and source
of cells. It is within the skill of the ordinary artisan to
determine the optimal density for a given set of culture conditions
and cells,
[0112] Also, effective atmospheric oxygen concentrations of less
than about 10%, including about 1-5% and, especially, 3-5%, can be
used at any time during the isolation, growth and differentiation
of MAPCs in culture.
[0113] Cells may be cultured under various serum concentrations,
e.g., about 2-20%. Fetal bovine serum may he used. Higher serum may
be used in combination with lower oxygen tensions, for example,
about 15-20%. Cells need not be selected prior to adherence to
culture dishes, For example, after a ficoll gradient, cells can be
directly plated, e.g., 250,000-500,000/cm.sup.2. Adherent colonies
can be picked, possibly pooled, and expanded.
[0114] In one embodiment, used in the experimental procedures the
Examples, high serum (around 15-20%) and low oxygen (around 3-5%)
conditions were used for the cell culture. Specifically, adherent
cells from colonies were plated and passaged at densities of about
1700-2300 cells/cm.sup.2 in 18% serum and 3% oxygen (with PDGF and
EGF).
[0115] In an embodiment specific for MAPCs, supplements are
cellular factors or components that allow MAPCs to retain the
ability to differentiate into all three lineages. This may be
indicated by the expression of specific markers of the
undifferentiated state, MAPCs, for example, constitutively express
Oct 3/4 (Oct 3A) and maintain high levels of telomerase.
Cell Culture
[0116] In general, cells useful for the invention can be maintained
and expanded in culture medium that is available to and well-known
in the art. Such media include, but are not limited to Dulbecco's
Modified Eagle's Medium.RTM. (DMEM), DMEM F12 medium.RTM., Eagle's
Minimum Essential Medium.RTM., F-12K medium.RTM., Iscove's Modified
Dulbecco's Medium.RTM. and RPMI-1640 medium.RTM.. Many media are
also available as a low-glucose formulations, with or without
sodium pyruvate.
[0117] Also contemplated is supplementation of cell culture medium
with mammalian sera. Sera often contain cellular factors and
components that are necessary for viability and expansion. Examples
of sera include fetal bovine serum (FBS), bovine serum (BS), calf
serum (CS), fetal calf serum (FCS), newborn calf serum (NCS), goat
serum (GS), horse serum (HS), human serum, chicken serum, porcine
serum, sheep serum, rabbit serum, serum replacements, and bovine
embryonic It is understood that sera can be heat-inactivated at
55-65.degree. C. if deemed necessary to inactivate components of
the complement cascade.
[0118] Additional supplements can also be used advantageously to
supply the cells with the necessary trace elements for optimal
growth and expansion. Such supplements include insulin,
transferrin, sodium selenium and combinations thereof. These
components can be included in a salt solution such as, but not
limited to Hanks' Balanced Salt Solution.RTM. (HBSS), Earle's Salt
Solution.RTM., antioxidant supplements, MCDB-201.RTM. supplements,
phosphate buffered saline (PBS), ascorbic acid and ascorbic
acid-2-phosphate, as well as additional amino acids. Many cell
culture media already contain amino acids, however some require
supplementation prior to culturing cells. Such amino acids include,
but are not limited to, L-alanine, L-arginine, L-aspartic acid,
L-asparagine, L-cysteine, L-cystine, L-glutamic acid, L-glutamine,
L-glycine, L-leucine, L-tysine, L-methionine, L-phenylalanine,
L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and
L-valine. It is well within the skill of one in the art to
determine the proper concentrations of these supplements.
[0119] Hormones can also be advantageously used in cell culture and
include, but are not limited to D-aldosterone, diethylstilbestrol
(DES), dexamethasone, .beta.-estradiol, hydrocortisone, insulin,
prolactin, progesterone, somatostatin/human growth hormone (HGH),
thyrotropin, thyroxine, and L-thyronine.
[0120] Lipids and lipid carriers can also be used to supplement
cell culture media, depending on the type of cell and the fate of
the differentiated cell Such lipids and carriers can include, but
are riot limited to cyclodextrin (.alpha., .beta., .gamma.),
cholesterol, linoleic acid conjugated to albumin, linoleic acid and
oleic acid conjugated to albumin, unconjugated linoleic acid,
linoleic-oleic-arachidonic acid conjugated to albumin, oleic acid
unconjugated and conjugated to albumin, among others.
[0121] Also contemplated is the use of feeder cell layers. Feeder
cells are used to support the growth of fastidious cultured cells,
particularly ES cells, Feeder cells are normal cells that have been
inactivated by .gamma.-irradiation. In culture, the feeder layer
serves as a basal layer for other cells and supplies cellular
factors without further growth or division of their own (Lim, J. W.
and Bodnar, A., 2002). Examples of feeder layer cells are typically
human diploid lung cells, mouse embryonic fibroblasts, Swiss mouse
embryonic fibroblasts, but can be any post-mitotic cell that is
capable of supplying cellular components and factors that are
advantageous in allowing optimal growth, viability, and expansion
of stem cells. In many cases, feeder cell layers are not necessary
to keep the ES cells in an undifferentiated, proliferative state,
as leukemia inhibitory factor (LIF) has anti-differentiation
properties. Therefore, supplementation with LIF could be used to
maintain MAPC in some species in an undifferentiated state.
[0122] Cells may be cultured in low-serum or serum-free culture
medium. Serum-free medium used to culture MAPCs is described in
U.S. Pat. No. 7,015,037. Many cells have been grown in serum-free
or low-serum medium. In this case, the medium is supplemented with
one or more growth factors. Commonly-used growth factors include
but are not limited to bone morphogenic protein, basis fibroblast
growth factor, platelet-derived growth factor, and epidermal growth
factor. See, for example, U.S. Pat. Nos. 7,169,610; 7,109,032;
7,037,721; 6,617,161; 6,617,159; 6,372,210; 6,224,860; 6,037,174;
5,908,782; 5;766,951; 5,397,706; and 4,657,866; all incorporated by
reference for teaching growing cells in serum-free medium.
[0123] Cells in culture can be maintained either in suspension or
attached to a solid support, such as extracellular matrix
components. Stem cells often require additional factors that
encourage their attachment to a solid support, such as type I and
type II collagen, chondroitin sulfate, fibronectin,
"superfibronectin" and fibronectin-like polymers, gelatin, poly-D
and poly-L-lysine, thrombospondin and vitronectin. One embodiment
of the present invention utilizes fibronectin. See, for example,
Ohashi et al., Nature Medicine, 13:880-885 (2007); Matsumoto et
al., J Bioscience and Bioengineering, 105:350-354 (2008); Kirouac
et al., Cell Stem Cell, 3:369-381 (2008); Chua et al.,
Biomaterials, 26:2537-2547 (2005); Drobinskaya et al. Stem Cells,
26:2245-2256 (2008); Dvir-Ginzberg et al., FASEB J, 22:1440-1449
(2008); Turner et al., J Biomed Mater Res Part B: Appl Biomater,
82B:156-168 (2007); and Miyazawa et al., Journal of
Gastroenterology and Hepatology, 22:1959-1964 (2007)).
[0124] Cells may also be grown in "3D" (aggregated) cultures. An
example is U.S. Provisional Patent Application No. 61/022,121,
filed Jan. 18, 2008.
[0125] Once established in culture, cells can be used fresh or
frozen and stored as frozen stocks, using, for example, DMEM with
40% FCS and 10% DMSO, Other methods for preparing frozen stocks for
cultured cells are also available to those of skill in the art.
Pharmaceutical Formulations
[0126] In certain embodiments, the purified cell populations are
present within a composition adapted for and suitable for delivery,
i.e., physiologically compatible. Accordingly, compositions of the
stem cell populations will often further comprise one or more
buffers (e.g., neutral buffered saline or phosphate buffered
saline), carbohydrates (e.g., glucose, mannose, sucrose or
dextrans), mannitol, proteins, polypeptides or amino acids such as
glycine, antioxidants, bacteriostats, chelating agents such as EDTA
or glutathione, adjuvants (e.g., aluminum hydroxide), solutes that
render the formulation isotonic, hypotonic or weakly hypertonic
with the blood of a recipient, suspending agents, thickening agents
and/or preservatives.
[0127] In other embodiments, the purified cell populations are
present within a composition adapted for or suitable for freezing
or storage.
[0128] In many embodiments the purity of the cells (or conditioned
medium) for administration to a subject is about 100%. In other
embodiments it is 95% to 100%. In some embodiments it is 85% to
95%, Particularly in the case of admixtures with other cells, the
percentage can be about 10%45%, 15%-20%, 20%-25%, 25%-30%, 30%-35%,
35%-40%, 40%45%, 45%-50%, 60%-70%, 78%-80%, 80%-90%, or 90%-95%. Or
isolation/purity can he expressed in terms of cell doublings where
the cells have undergone, for example, 10-20, 20-30, 30-40, 40-50
or more cell doublings.
[0129] The numbers of cells in a given volume can he determined by
well known and routine procedures and instrumentation. The
percentage of the cells in a given volume of a mixture of cells can
be determined by much the same procedures. Cells can be readily
counted manually or by using an automatic cell counter. Specific
cells can he determined in a given volume using specific staining
and visual examination and by automated methods using specific
binding reagent, typically antibodies, fluorescent. tags, and a
fluorescence activated cell sorter.
[0130] The choice of formulation for administering the cells for a
given application will depend on a variety of factors. Prominent
among these will be the species of subject, the nature of the
disorder, dysfunction, or disease being treated and its state and
distribution in the subject, the nature of other therapies and
agents that are being administered, the optimum route for
administration, survivability via the route, the dosing regimen,
and other factors that will be apparent to those skilled in the
art, In particular, for instance, the choice of suitable carriers
and other additives will depend on the exact route of
administration and the nature of the particular dosage form.
[0131] For example, cell survival can be an important determinant
of the efficacy of cell-based therapies. This is true for both
primary and adjunctive therapies. Another concern arises when
target sites are inhospitable to cell seeding and cell growth. This
may impede access to the site and/or engraftment there of
therapeutic cells. Various embodiments of the invention comprise
measures to increase cell survival and/or to overcome problems
posed by barriers to seeding and/or growth.
[0132] Final formulations of the aqueous suspension of cells/medium
will typically involve adjusting the ionic strength of the
suspension to isotonicity (i.e., about 0.1 to 0.2) and to
physiological. pH (i.e., about pH 6.8 to 7.5). The final
formulation will also typically contain a fluid lubricant, such as
maltose, which must be tolerated by the body. Exemplary lubricant
components include glycerol, glycogen, maltose and the like.
Organic polymer base materials, such as polyethylene glycol and
hyaluronic acid as well as non-fibrillar collagen, preferably
succinylated collagen, can also act as lubricants. Such lubricants
are generally used to improve the injectability, intrudability and
dispersion of the injected biomaterial at the site of injection and
to decrease the amount of spiking by modifying the viscosity of the
compositions. This final formulation is by definition the cells in
a pharmaceutically acceptable carrier.
[0133] The cells are subsequently placed in a syringe or other
injection apparatus for precise placement at the site of the tissue
defect. The term "injectable" means the formulation can he
dispensed from syringes having a gauge as low as 2.5 under normal
conditions under normal pressure without substantial spiking.
Spiking can cause the composition to ooze from the syringe rather
than be injected into the tissue. For this precise placement,
needles as fine as 27 gauge (200.mu. I.D.) or even 30 gauge
(150.mu. I.D.) are desirable. The maximum particle size that can be
extruded through such needles be a complex function of at least the
following: particle maximum dimension, particle aspect ratio
(length:width) particle rigidity, surface roughness of particles
and related factors affecting particle:particle adhesion, the
viscoelastic properties of the suspending fluid, and the rate of
flow through the needle. Rigid spherical beads suspended in a
Newtonian fluid represent the simplest case, while fibrous or
branched particles in a viscoelastic fluid are likely to be more
complex,
[0134] The desired isotonicity of the compositions of this
invention may be accomplished using sodium chloride, or other
pharmaceutically acceptable agents such as dextrose, boric acid,
sodium tartrate, propylene glycol, or other inorganic or organic
solutes. Sodium chloride is preferred particularly for buffers
containing sodium ions.
[0135] Viscosity of the compositions, if desired, can be maintained
at the selected level using a pharmaceutically acceptable
thickening agent, Methylcellulose is preferred because it is
readily and economically available and is easy to work with. Other
suitable thickening agents include, for example, xanthan gum,
carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the
like, The preferred concentration of the thickener will depend upon
the agent selected. The important point is to use an amount, which
will achieve the selected viscosity. Viscous compositions are
normally prepared front solutions by the addition of such
thickening agents.
[0136] A pharmaceutically acceptable preservative or stabilizer can
be employed to increase the life of cell/medium compositions, if
such preservatives are included, it is well within the purview of
the skilled artisan to select compositions that will not affect the
viability or efficacy of the cells,
[0137] Those skilled in the art will recognize that the components
of the compositions should be chemically inert. This will present
no problem to these skilled in chemical and pharmaceutical
principles. Problems can he readily avoided by reference to
standard texts or by simple experiments (not involving undue
experimentation) using information provided by the disclosure, the
documents cited herein, and generally available in the art.
[0138] Sterile injectable solutions can be prepared by
incorporating the cells/medium utilized in practicing the present,
invention in the required amount of the appropriate solvent with
various amounts of the other ingredients, as desired.
[0139] In some embodiments, cells/medium are formulated in a unit
dosage injectable form, such as a solution, suspension, or
emulsion. Pharmaceutical formulations suitable for injection of
cells/medium typically are sterile aqueous solutions and
dispersions. Carriers for injectable formulations can be a solvent
or dispersing medium containing, for example, water, saline,
phosphate buffered saline, polyol (for example, glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof.
[0140] The skilled artisan can readily determine the amount of
cells and optional additives, vehicles, and/or carrier in
compositions to be administered in methods of the invention.
Typically, any additives (in addition to the cells) are present in
an amount of 0.001 to 50 wt % in solution, such as in phosphate
buffered saline. The active ingredient is present in the order of
micrograms to milligrams, such as about 0.0001 to about 5 wt %,
preferably about 0.0001 to about 1 wt %, most preferably about
0.0001 to about 0.05 wt %, or about 0.001 to about 20 wt %,
preferably about 0.01 to about 10 wt %, and most preferably about
0.05 to about 5 wt %.
[0141] In some embodiments cells are encapsulated for
administration, particularly where encapsulation enhances the
effectiveness of the therapy, or provides advantages in handling
and/or shelf life. Encapsulation in some embodiments where it
increases the efficacy of cell mediated immunosuppression may, as a
result, also reduce the need for immunosuppressive drug
therapy.
[0142] Also, encapsulation in some embodiments provides a barrier
to a subject's immune system that may further reduce a subject's
immune response to the cells (which generally are not immunogenic
or are only weakly immunogenic in allogeneic transplants), thereby
reducing any graft rejection or inflammation that might occur upon
administration of the cells.
[0143] Cells may be encapsulated by membranes, as well as capsules,
prior to implantation. It is contemplated that any of the many
methods of cell encapsulation available may be employed. In some
embodiments, cells are individually encapsulated. In some
embodiments, many cells are encapsulated within the seine membrane.
In embodiments in which the cells are to be removed following
implantation, a relatively large size structure encapsulating many
cells, such as within a single membrane, may provide a convenient
means for retrieval.
[0144] A wide variety of materials may be used in various
embodiments for microencapsulation of cells. Such materials
include, for example, polymer capsules,
alginate-poly-L-lysine-alginate microcapsules, barium poly-L-lysine
alginate capsules, barium alginate capsules,
polyacrylonitrile/polyvinylchloride (PAN/PVC) hollow fibers, and
polyethersulfone (PES) hollow fibers.
[0145] Techniques for microencapsulation of cells that may be used
for administration of cells are known to those of skill in the art
and are described, for example, in Chang, P., et al,, 1999;
Matthew, H. W., et al., 1991; Yanagi, K., et al., 1989; Cai Z. H.,
et al., 1988; Chang, T. M., 1992 and in U.S. Pat. No. 5,639,275
(which, for example, describes a biocompatible capsule for
long-term maintenance of cells that stably express biologically
active molecules. Additional methods of encapsulation are in
European Patent Publication No. 301,777 and U.S. Pat Nos.
4,353,888; 4,744,933; 4,749,620; 4,814,274; 5,084,350; 5,089,272;
5,578,442; 5,639,275; and 5,676,943. All of the foregoing are
incorporated herein by reference in parts pertinent to
encapsulation of cells.
[0146] Certain embodiments incorporate cells into a polymer, such
as a biopolymer or synthetic polymer. Examples of biopolymers
include, but are not limited to, fibronectin, fibin, fibrinogen,
thrombin, collagen, and proteoglycans. Other factors, such as the
cytokines discussed above, can also be incorporated into the
polymer. In other embodiments of the invention, cells may be
incorporated in the interstices of a three-dimensional gel. A large
polymer or gel, typically, will be surgically implanted. A polymer
or gel that can be formulated in small enough particles or fibers
can be administered by other common, more convenient, non-surgical
routes.
Dosing
[0147] Compositions can be administered in dosages and by
techniques well known to those skilled in the medical and
veterinary arts taking into consideration such factors as the age,
sex, weight, and condition of the particular patient, and the
formulation that will be administered (e.g., solid vs. liquid).
Doses for humans or other mammals can be determined without undue
experimentation by the skilled artisan, from this disclosure, the
documents cited herein, and the knowledge in the art,
[0148] The dose of cells/medium appropriate to be used in
accordance with various embodiments of the invention will depend on
numerous factors. it may vary considerably for different
circumstances. The parameters that will determine optimal doses to
be administered for primary and adjunctive therapy generally will
include some or all of the following: the disease being treated and
its stage; the species of the subject, their health, gender, age,
weight, and metabolic rate; the subject's immunocompetence; other
therapies being administered; and expected potential complications
from the subject's history or genotype. The parameters may also
include: whether the cells are syngeneic, autologous, allogeneic,
or xenogeneic; they potency (specific activity); the site and/or
distribution that must be targeted for the cells/medium to be
effective; and such characteristics of the site such as
accessibility to cells/medium and/or engraftment of cells.
Additional parameters include co-administration with other factors
(such as growth factors and cytokines). The optimal dose in a given
situation also will take into consideration the way in which the
cells/medium are formulated, the way they are administered, and the
degree to which the cells/medium will be localized at the target
sites following administration. Finally, the determination of
optimal dosing necessarily will provide an effective dose that is
neither below the threshold of maximal beneficial effect nor above
the threshold where the deleterious effects associated with the
dose outweighs the advantages of the increased dose.
[0149] The optimal dose of cells for some embodiments will be in
the range of doses used for autologous, mononuclear bone marrow
transplantation. For fairly pure preparations of cells, optimal
doses in various embodiments will range from 10.sup.4 to 10.sup.8
cells/kg of recipient mass per administration, In some embodiments
the optimal dose per administration will be between 10.sup.5 to
10.sup.7 cells/kg. In many embodiments the optimal dose per
administration will be 5.times.10.sup.5 to 5.times.10.sup.6
cells/kg. By way of reference, higher doses in the foregoing are
analogous to the doses of nucleated cells used in autologous
mononuclear bone marrow transplantation. Some of the lower doses
are analogous to the number of CD34.sup.+ cells/kg used in
autologous mononuclear bone marrow transplantation.
[0150] It is to be appreciated that a single dose may be delivered
all at once, fractionally, or continuously over a period of time.
The entire dose also may be delivered to a single location or
spread fractionally over several locations.
[0151] In various embodiments, cells/medium may be administered, in
an initial dose, and thereafter maintained by further
administration. Cells/medium may be administered by one method
initially, and thereafter administered by the same method or one or
more different methods. The levels can be maintained by the ongoing
administration of the cells/medium. Various embodiments administer
the cells/medium either initially or to maintain their level in the
subject or both by intravenous injection. In a variety of
embodiments, other forms of administration, are used, dependent
upon the patients condition and other factors, discussed elsewhere
herein.
[0152] It is noted that human subjects are treated generally longer
than experimental animals; but, treatment generally has a length
proportional to the length of the disease process and the
effectiveness of the treatment, Those skilled in the art will take
this into account in using the results of other procedures carried
out in humans and/or in animals, such as rats, mice, non-human
primates, and the like, to determine appropriate doses for humans.
Such determinations, based on these considerations and taking into
account guidance provided by the present disclosure and the prior
311 will enable the skilled artisan to do so without undue
experimentation.
[0153] Suitable regimens for initial administration and further
doses or for sequential administrations may all be the same or may
be variable. Appropriate regimens can be ascertained by the skilled
artisan, from this disclosure, the documents cited herein, and the
knowledge in the art.
[0154] The dose, frequency, and duration of treatment will depend
on many factors, including the nature of the disease, the subject,
and other therapies that may be administered. Accordingly, a wide
variety of regimens may be used to administer the cells/medium.
[0155] In some embodiments cells/medium are administered to a
subject in one dose. In others. cells/medium are administered to a
subject in a series of two or more doses in succession. In some
other embodiments wherein cells/medium are administered in a single
dose, in two doses, and/or more than two doses, the doses may be
the same or different, and they are administered with equal or with
unequal intervals between them.
[0156] Cells/medium may be administered in many frequencies over a
wide range of times. in some embodiments, they are administered
over a period of less than one day. In other embodiment they are
administered over two, three, four, five, or six days. In some
embodiments they are administered one or more times per week, over
a period of weeks. In other embodiments they are administered over
a period of weeks for one to several months. In various embodiments
they may be administered over a period of months. In others they
may be administered over a period of one or more years. Generally
lengths of treatment will be proportional to the length of the
disease process, the effectiveness of the therapies being applied,
and the condition and response of the subject being treated.
[0157] For example, for spinal cord injury it is explained in this
document that there may be two phases. In the animal models, in the
first phase, macrophages do not infiltrate the lesion and this
lasts for about 24 hours. In the second phase, however, macrophages
do infiltrate the lesion and this sequence of events may be taken
into consideration when assessing treatment regimens. In one
embodiment, cells are administered even during the first phase,
such as immediately after injury or as close to the injury as
possible in anticipation of the infiltration of macrophages or
other cells that would interact with dystrophic axons. Treatment
may then be continued to coincide with initial and further
infiltration of macrophages and may be preventatively continued or
possibly discontinued when it is determined that macrophages or the
other relevant cells are no longer infiltrating the injury.
EXAMPLES
Example I
[0158] "Glial Scar Model" Aggrecan-laminin opposing spot gradients
(Tom et al., 2004; Steinmetz et al., 2005). These references are
incorporated by reference for teaching the glial scar model. This
model provides an assay for the effectiveness of cells, proteins,
medium, etc., in reducing adhesion/retraction in vitro.
[0159] PGs can induce the so-called dystrophic state in axons if
the inhibitory matrix is presented in a spatial organization that
more closely resembles that which develops after lesions in vivo.
To do this, spots of a solution of the PG aggrecan and the
growth-promoting molecule laminin were placed on nitrocellulose
coverslips and air dried.
[0160] A consistent artifact of drying produced a crude gradient in
which the rim of the spot contained an increasingly higher
concentration of aggrecan than in the center. The very outermost
part of the rim contained a lower concentration of laminin than any
more central region, The optimal ECM concentrations (0.7 mg/ml
aggrecan and 5 .mu.g/ml laminin) resulted in good cell attachment.
Thus, the high aggrecan-low laminin outer rim appeared to be a
particularly harsh terrain for regenerating neurites, None entered
inward into the spot from the laminin surround by crossing its
sharp outer interface. Fibers growing centripetally from within the
center of the spot were able to enter the inner portion of the rim
but could grow no farther. Once within the gradient, axons appeared
trapped. Club-like, dystrophic endballs formed at the ends of
neurites within the gradient. To observe the behavior of the
"dystrophic endings" time-lapse microscopy was used. Dystrophic
growth cones often managed to advance short distances, but
inevitably, the struggling growth cone would round up into a more
compact ball and retract, only to start moving again.
Example II
Axonal Retraction and Microphages
Summary
[0161] In vivo, a close correlation was sound between dystrophic
retraction clubs at the ends of severed axons and ED-1.sup.+ cells
following a dorsal column crush spinal cord injury (FIG. 3). The in
vitro model of the glial scar (Tom et al., 2004; Steinmetz et al.,
2005) was applied to examine the interactions between axons and
ED-1.sup.+ cells in real time. Direct cell-cell contact between
dystrophic growth cones and ED-1.sup.+ macrophages induced a long
distance axonal retraction (FIGS. 4, 5). The result of using
clodronate liposomes (Popovich, et al., 1999) for macrophage
depletion in viva was significant reduction in axonal retraction in
the clodronate-treated animals compared to controls. These data
indicate that ED-1.sup.+ cells are directly responsible for
retraction of injured spinal cord axons through physical cell-cell
interactions.
Results
1. Ascending Dorsal Column Sensory Axons Retract Extensively
Following Spinal Cord Injury
[0162] It was considered by the inventors that infiltration of
activated macrophages could play a direct role in axonal
retraction. Within sub-acute and chronic. spinal cord lesions a
close association was found between activated macrophages and the
ends of regenerating axons, allowing for the possibility of direct
physical interactions between these two cell types. The extent of
sensory axon retraction after dorsal column crush injury was then
characterized and correlated with the infiltration of macrophages
into the lesion. The dorsal columns of adult female Sprague-Dawley
rats were crushed at the level of C8 and a subpopulation of injured
neurons were traced via dextran-Texas Red labeling of the sciatic
nerve. Spinal cord tissue was harvested at 2, 4, 7, 14, and 28 days
post-lesion and the distances between the ends of the labeled
fibers and the center of the lesions were measured. By 2 days
post-lesion, axons had already retracted an average distance of
343.+-.46.92 um (mean.+-.SD), however, this early retraction was
most likely due to intrinsic mechanisms within the neurons
themselves (Kerschensteiner et al., 2005). It is important to note
that at 2 days post injury the lesion was composed of mainly
reactive astrocytes (GFAP.sup.+) and a few ED-1.sup.+ cells, Which
were most likely resident microglia. Between days 2 and 7
post-lesion there was a dramatic increase in the number of
ED-1.sup.+ cells within the lesion, the vast majority of which were
most likely infiltrating macrophages (Popovich at al., 1997;
Donnelly and Popovich, 2007). The second phase of retraction of
ascending sensory fibers within the dorsal columns occurred most
rapidly over the first week and then progressively over the next
few weeks. By twenty-eight days post-lesion the axons had retracted
to an average distance of 774.+-.70.26 um from the lesion
epicenter. These data show that the timing of ascending sensory
axon retraction corresponds spatiotemporally with the infiltration
and accumulation of ED-1.sup.+ cells within the lesion.
2. Depletion of Activated Macrophages Reduces Axonal Retraction in
vivo
[0163] The majority of the second phase of axonal retraction in
vivo occurs in the ascending dorsal column sensory axons between
two and seven days post-lesion corresponding temporally to
macrophage infiltration. in order to further implicate macrophages
in axonal retraction in vivo animals were treated with clodronate
liposomes in order to deplete circulating monocytes/'macrophages
(van Rooijen et al., 1997; Popovich at al., 1999). Animals received
injections of clodronate Liposomes every other day starting
treatment one day prior to injury to deplete circulating
monocytes/macrophages. Animals were then assessed for axonal
retraction at 2 d, 4 d, and 7 d post-lesion (FIG. 3). Animals that
received injections of clodronate liposomes displayed a significant
reduction of retraction at 4 d and 7d post-lesion (402.+-.81.85 um
and 439.+-.46.33 um, respectively) as compared to those receiving
control liposomes at 4 d and 7 d post-lesion (586.+-.42.89 um and
806.+-.62.71 um respectively). The reduction in retraction
correlated with significantly reduced numbers of ED-1.sup.+ cells
within the lesion in clodronate-treated animals compared to empty
liposome controls. Clodronate liposome treatment also resulted in
an increase of GFAP.sup.+ astrocyte processes in the lesion core,
correlating with previous observations that macrophage depletion
leads to a decrease in cavitation (Popovich et al., 1999).
Importantly, there was no difference in the amount of retraction
exhibited in the clodronate-treated and control liposome-treated
animals at 2 d post-lesion. Macrophage infiltration has not yet
occurred at this time, indicating that that the first stage of
axonal retraction is macrophage-independent, and perhaps due to
endogenous neuronal mechanisms or, potentially, interactions with
activated resident microglia. Clodronate-mediated depletion of
circulating macrophages/monocytes did prevent axonal retraction
normally observed at 4 d and 7 d post-lesion, indicating that this
second phase of retraction was caused by the actions of
infiltrating macrophages. There was no evidence of significant
regeneration (i.e., axonal elongation beyond the center of the
lesion) in the clodronate-treated animals.
3. Dystrophic Growth Cones Retract Extensively Alter Contact with
Macrophages in an in vitro Model of the Glial Scar
[0164] The observation of the close association of ED-1.sup.+ cells
with injured axons in vivo suggested that interactions between
these cell types may play a role in axonal retraction. The
interactions between adult sensory neuron axons and macrophages in
an in vitro model of the glial scar were studied. Following a
30-minute period of baseline observation, NR 8383 macrophages were
added to the cultures and their interactions with dystrophic axons
monitored. Direct cell-cell contacts were frequently observed
between dystrophic axons and macrophages. These contacts were of an
extended duration and, when coupled with migration of the
macrophage, led to dramatic manipulations of the axon that resulted
in forceful bending and lifting of the axon from the substrate. It
was evident that strong, long-lasting adhesions could be made
between the two cell types since lengthy trailing processes
connecting the two cells often remained after the macrophages
migrated away from the axons. However, macrophage-induced
retraction did not permanently prevent re-growth of the axon, as
some axons that lost macrophage contact following retraction were
able to extend until they again became dystrophic. Direct cell-cell
contact between these two cell types eventually led to extensive
retraction of the axon 100% of the time. Therefore, macrophage
contact induced retraction of dystrophic axons in an in vitro model
of the glial sear.
4. Direct Physical Cell-Cell Interactions are Required for
Macrophage-Induced Retraction
[0165] There were numerous instances in which macrophages were
observed to migrate very close to, but not touch, dystrophic axons
and axonal retraction was not observed in these cases. To determine
whether physical interactions between axons and macrophages were
required to induce axonal retraction or if macrophage-derived
factors were sufficient, macrophages were treated with trypsin to
remove extracellular proteins prior to their addition to the DRG
cultures. Pre-treatment of macrophages with trypsin still allowed
for extensive macrophage mobility and multiple collisions with
axons. However, the treatment completely prevented the macrophages
from physically tethering to the axons, and consequently no
retraction was observed in the absence of long lasting direct
cell-cell contacts. It was possible, however, that macrophages were
secreting a factor(s) that induced retraction. To test this
hypothesis macrophage-conditioned media was added to dystrophic
axons in vitro. Macrophage-conditioned media did not induce
retraction. Therefore, the mere presence of macrophages or their
secreted factors in the vicinity of axons were not capable of
inducing axonal retraction in the absence of physical interactions
with dystrophic axons.
5. Role of the Substrate in Macrophage-Induced Retraction
[0166] To determine whether the substrate plays a role in axonal
retraction, adult sensory neurons were cultured on a uniform
growth-promoting laminin substrate that does not produce dystrophic
growth cones, Growth cones on laminin were flattened with numerous
filopodia and lamellapodia and overall axon extension occurred at a
constant rate. When macrophages were added to these cultures,
direct cell-cell contact with axons was observed. However, these
contacts were transient and quickly broken, not as extensive, and
did not result in axonal retraction. Remnants of membrane contact
points were reabsorbed rapidly hack into the neurons, and the
growth cones continued to extend across the substrate unimpeded.
Therefore, macrophage-induced axonal retraction was
substrate-dependent; neurons in an active growth state On the
permissive substrate laminin were not susceptible to macrophage
contact unlike those in a state of dystrophy induced by a CSPG
gradient.
6. Activated Primary Macrophages also Induce Axonal Retraction
[0167] A further issue was if primary macrophages interacted with
dystrophic awns In the same manner as the NR 8383 macrophage cell
line in vitro. Progenitor cells from the bone marrow of adult
Sprague-Dawley rats were harvested and differentiated. into
macrophages in vitro, yielding a culture of greater than 80%
ED-1.sup.+ cells. This particular population of macrophages has
been shown to retain the phenotypic, morphological and functional
characteristics of macrophages found in spinal cord lesions unlike
populations harvested from other bodily sources (Longbrake et al.,
2007). The ability of primary macrophages to induce axonal
retraction was then assayed. Un-stimulated primary macrophages were
not capable of inducing retraction. When added to the spot gradient
neuronal culture, these macrophages adhered to the substrate but
were not motile, displaying characteristics of macrophages in a
resting state. Contacts with axons occurred only when macrophages
settled directly onto dystrophic axons. Neither the macrophages nor
their cell-cell interactions exhibited any of the physical
characteristics previously observed with the cell line macrophages,
i.e. no tugging, no signs of physical attachment via cell
processes, etc.
[0168] It was possible that macrophages must be in an activated
state in order to interact with dystrophic axons. Primary
macrophages were stimulated with the activating cytokine
interferon-gamma in culture prior to addition to the time-lapse
culture dishes. While these macrophages exhibited a moderate state
of activation and a slightly rounded morphology, they were still
not motile and did not form strong attachments with dystrophic
axons and, consequently did not induce axonal retraction. The
primary macrophages were further stimulated with a combination of
interferon-gamma and lipopolysaccharide (LPS) prior to addition to
the DRG cultures. These macrophages displayed the morphology and
behavior of activated macrophages: rounded, phagocytic shape and
highly motile. These activated macrophages induced retraction of
dystrophic, axons as frequently as cell line macrophages. They
displayed vigorous physical interactions with dystrophic axons,
resulting in strong adhesions between cells and physical grasping,
tugging and lifting of axons from the substrate. Primary
macrophages, when in an activated state, induced retraction of
dystrophic axons in vitro validating the use of cell line
macrophages in this study of axonal retraction in vitro. Therefore,
the majority of the experiments were carried out with the NR8383
macrophage cell line because it constituted a pure population of
cells that were in a constant state of activation, similar to
macrophages found within spinal cord lesions without additional
stimulation.
7. Activated are Moderately Capable of Inducing Axonal Reaction in
vitro
[0169] While macrophages do not typically invade the injured spinal
cord until three days post-lesion, resident microglia within the
CNS respond to injury immediately (Watanabe et al., 1999).
Microglia within a lesion become activated and phagocytic, much
like activated macrophages. A limited number of ED-1.sup.+ cells
was found within the lesion at 2 days post-injury, before typical
macrophage infiltration, which were most likely resident microglia.
The potential contribution of microglia to axonal retraction was
assessed using the in vitro model. Cortical microglia were
harvested from P1 Sprague-Dawley rats and matured in vitro prior to
their addition to time-lapse cultures. Similar to primary
macrophages, primary microglia had to be stimulated with
interferon-gamma and LPS to become activated in culture.
Un-stimulated microglia failed to adhere to the laminin/aggrecan
spot gradient substrate, which prevented them from interacting with
dystrophic axons in our model. However, stimulated microglia did
adhere and physically interact with axons, inducing retraction 50%
of the time, however, the contacts between activated microglia and
dystrophic. axons were not as strong as those of macrophages.
Therefore, microglia activated experimentally can also play a role
in the induction of axonal retraction.
8. Astrocytes, Another CNS Cell Type, Fail to Induce Axonal
Retraction in vitro
[0170] Another question was if the induction of retraction of
dystrophic axons in vitro is specific to phagocytic cell types
normally found within a lesioned spinal cord and not merely the
result of the interactions of dystrophic axons with any other cell
type. Astrocytes are an integral component of the glial scar
following injury to the CNS. They are present in high numbers and
extensively contact regenerating axons. Cortical astrocytes were
allowed to mature in vitro before addition to DRG cultures.
Astrocytes adhered to the substrate and contacted dystrophic axons
extensively. Once bound to the substrate, astrocytes migrated
rapidly down the aggrecan gradient, away from the rim. Astrocyte
processes spread out over axons, sometimes resulting in lateral
displacement of the axon. However, these contacts did not lead to
retraction of the contacted axon. Therefore, the induction of
retraction was specific to interactions with ED-1.sup.+ phagocytic
cells and not merely physical interactions with any other cell
type.
Materials and Methods
1. Dorsal Column Crush Lesion Model
[0171] Thirty-three adult female Sprague-Dawley rats (250-300 g)
were used for in vivo studies. Rats were anesthetized with inhaled
isofluorane gas (2%) for all surgical procedures. A T1 laminectomy
was performed to expose the dorsal aspect of the C8 spinal cord
segment. A durotomy was made bilaterally 0.75 mm from midline with
a 30 gauge needle. A dorsal column crush lesion was then made by
inserting Dumont # 3 jeweler's forceps into the dorsal spinal cord
at to a depth of 1.0 mm and squeezing the forceps, holding pressure
for ten seconds and repeated two additional times. Completion of
the lesion was verified by observation of white matter clearing.
The holes in the dura were then covered with gel film. The muscle
layers were sutured with 4-0 nylon suture, and the skin was closed
with surgical staples. Upon closing of the incision, animate
received Marcaine (1.0 mg/kg) subcutaneously along the incision as
well as Buprenorphine (0.1 mg/kg) intramuscularly.
Post-operatively, animals were kept warm with a heating lamp during
recovery from anesthesia and allowed access to food and water ad
libitum. Animals were killed at 2, 4, 7, 14, or 28 days post-lesion
(N=3 per group). All animal procedures were carried out in
accordance with the guidelines and protocols of the Animal Resource
Center at Case Western Reserve University.
2. Macrophage Depletion
[0172] Animals received intraperitoneal injections of
liposome-encapsulated clodronate or empty liposome control on the
day before the dorsal column crush injury and also one day
post-lesion for the 2 d time-point, on days 1 and 3 post-lesion for
the 4 d timepoint, and on days 1, 3, and 5 post-lesion for the 7 d
timepoint (N=3 per group). Clodronate was a gift from Roche
Diagnostics GmbH, Mannheim, Germany. Clodronate was encapsulated in
liposomes are described earlier (Van Rooijen and Sanders,
1994).
3. Axon Labeling
[0173] Two days before sacrifice, the dorsal columns were labeled
unilaterally with Texas-Red conjugated 3000 MW dextran. Briefly,
the sciatic nerve of the right hindlimb was exposed and crushed
with Dumont #3 forceps for tens seconds and repeated two additional
times. 1.0 uL of 3000 MW dextran-Texas-Red 10% in sterile water was
the injected via a Hamilton syringe into the sciatic nerve at the
crush site. The muscle layers were closed with 4-0 nylon suture and
the skin with surgical staples. Upon closing of the incision,
animals received Marcaine (1.0 mg kg) subcutaneously along the
incision as well as Buprenorphine (0.1 mg/kg) intramuscularly.
Post-operatively, animals were kept warm with a heating lamp during
recovery from anesthesia and allowed access to food and water ad
libitum. Animals were killed two days following labeling with an
overdose of isofluorane and perfused with PBS followed by 4% PFA.
Tissue was harvested and post-fixed 4% PFA and processed for
immunohistochemistry.
4. Immunohistochemistry
[0174] Tissue was post fixed in 4% PFA overnight and then submersed
in 30% sucrose overnight, frozen in OCT mounting media, and cut on
a cryostat into 20 um longitudinal sections. Tissue was then
stained with anti-GFAP (Accurate Chemical and Scientific
Corporation, Westbury, N.Y.), anti-ED-1 (Millipore, Billerica,
Mass.) and incubated with Alexafluor-405 or Alexafluor-488
(Invitrogen, Carlsbad, Calif.) respectively, and then imaged on a
Zeiss Axiovert 510 laser-scanning confocal microscope.
5. In Vivo Axonal Retraction Quantification
[0175] Three consecutive sections starting at a depth of 200 um
below the dorsal surface of the spinal cord per animal were
analyzed per animal to quantify axonal retraction. The lesion
center was identified via characteristic GFAP and ED-1 staining
patterns and the distance between the end of the labeled axons and
the centered using Zeiss LSM 5 Image Browser software. The
measurements from all sections from all animals in a group were
averaged to yield the average distance of retraction per time
point.
[0176] The technique utilized to trace injured fibers labels axons
located very superficially within the dorsal columns, Also, the
numbers of fibers labeled can vary due to the extent of
fasciculation of the sciatic nerve at the level at which the tracer
is injected. Labeled axons were quantified at only that depth for
multiple reasons. This depth consistently contained labeled fibers
in all animals, whereas sonic animals did not have labeled fibers
at deeper depths. The linear extent of the lesion increases at
deeper levels of the dorsal columns. Therefore axons located deeper
within the spinal cord encounter a much larger lesion than those at
more superficial levels. Quantification of distances of retraction
must occur at similar locations of the lesion to allow for accurate
comparison between animals and groups. Quantification of the entire
population of labeled axons could lead to skewing of results due to
differences in the extent of labeling. Instead, a specific
population and location of labeled axons were quantified, so they
could be consistently examined and accurately quantified in all
animals
6. DRG Dissociation
[0177] DRGs were harvested as previously described (Tom et al.,
2004; Davies et al., 1999). Briefly, DRGs were dissected out of
adult female Sprague-Dawley rats (Zivic Miller, Harlan). Both the
central and peripheral roots were removed and ganglia incubated in
a solution of Collagenase II (200 U/mL, Worthington) and Dispase II
(2.5 U/mL, Roche) in MSS. The digested DRGs were rinsed and gently
triturated in fresh HBSS-CMF three times followed by low speed
centrifugation. The dissociated DRGs were then resuspended in
Neurobasal-A media supplemented with B-27, Glutamax, and
Pennicillin/Streptamycin (all from Invitrogen) and counted. DRGs
were plated on Delta-T dishes (Fisher,) at a density of 3,000
cells/mL for a total of 6,000 cells/dish.
7. Time-Lapse Dish Preparation
[0178] Delta-T cell culture dishes (Fisher, Pittsburgh, Pa.) were
prepared similarly to Tom et al., 2004. Briefly, a single hole was
drilled through the upper half of each dish with a number 2 bit to
create a port for the addition of cells, enzymes, inhibitors, etc.
to the cultures during time-lapse microscopy, Dishes were then
rinsed with sterile water and coated with poly-1-lysine (0.1 mg/mL,
Sigma) overnight at room temperature, rinsed with sterile water and
allowed to dry. Aggrecan gradient spots were created by pipetting
2.0 uL aggrecan solution (2.0 mg/mL, Sigma in HBSS-CMF, Invitrogen)
onto the culture surface and allowed to dry. Six spots were placed
per dish. After the aggrecan spots dried completely, the entire
surface of the dish was bathed in laminin solution (10 ug/mL, BTI,
Stoughton, Mass.) in HBSS-CMF for three hours at 37 degrees
Celsius. The laminin bath was then removed immediately before
plating of cells. Dishes containing a laminin only substrate were
prepared as above with only the laminin bath and no aggrecan. The
concentrations of substrates used here differ from those used by
Tom et al., 2004. The clarity of the microscopy can be improved by
removing the nitrocellulose from the dish preparation protocol.
However, to compensate for the difference in substrate binding to
the dish surface, the concentrations of the substrates used was
recalibrated to those listed above.
[0179] Following time-lapse imaging, DRGs were fixed in 4% PFA and
immunostained with anti B-tubulin-type III (1:500; Sigma, St.
Louis, Mo.) and anti-chondroitin sulfate (CS-56, 1:500, Sigma).
8. Cell Line Macrophage Cultures
[0180] NR8383 cells (ATCC # CRL-2192) an adult Sprague-Dawley
alveolar macrophage cell line were cultured as described by Yin at
al. (2003). Briefly, cells were cultured in uncoated tissue culture
flasks (Corning) in F-12K media (Invitrogen) supplemented with 15%
FBS, Glutamax, Penn/Strep (Invitrogen), and sodium bicarbonate
(Sigma) and fed two to three times per week. This cell line formed
a mixed culture of adherent and suspended cells and was passed by
collecting and replating floating cells at the time of feeding. To
prepare the cell line macrophages for time-lapse microscopy
experiments, cells were harvested with 0.5% trypsin/EDTA (Sigma)
washed three times with serum-free F-12K, and plated in uncoated
tissue culture flasks at a density of 1.0.times.10.sup.6/mL in
serum free F-12K. Prior to use in time-lapse experiments the
following day, the cultured cell line macrophages were harvested
with EDTA and a cell scraper and resuspended in Neurobasal-A
supplemented as above with the addition of HEPES (50 uM, Sigma) at
a density of 2.5.times.30.sup.5/70 uL.
9. Primary Bone Marrow-Derived Macrophage Cultures
[0181] Bone marrow progenitor cells were harvested based on
previously established protocol (Tobian et al., 2004). Briefly,
femurs were removed from adult female Sprague-Dawley rats
(225-275g, Harlan). The ends of the femurs were removed, a syringe
containing cold DMEM supplemented with 10% FBS, Glutamax,
Penn/Strep, beta-mercaptoethanol, and HEPES (Invitrogen) (D10F) was
inserted into the femur and the hone marrow was flushed out and
collected. The resulting cell mixture was then passed through a 70
micron filter and centrifuged. Supernatant was then removed, the
resulting cell pellet resuspended in AKT lysing buffer
(BioWhitacre) to lyse red blood cells, and centrifuged. The
supernatant was removed and the pellet containing bone marrow
precursor cells was resuspended and plated in DMEM above
additionally supplemented with 20% LADMAC cell line-conditioned
media (generous gift of Dr. Clifford Harding) to induce
differentiation into macrophages. Cells were fed on days 5, 7, 9,
and harvested for experimentation on day 10 in culture. One day
prior to time-lapse experiments, primary macrophages were harvested
with trypsin/EDTA, washed three times with D10F, and plated in
uncoated petri dishes (Falcon) in D10F at a density of
1.0.times.10.sup.6/mL. The following day, the primary macrophages
were harvested with EDTA wad a cell scraper and resuspended in
Neurobasal-A plus HEPES at a density of 5.0.times.10.sup.5/70 uL
for time-lapse microscopy experiments.
10. Cortical Astrocyte Preparation
[0182] Cortical astrocytes were collected by removing the cortices
of a P0-P1 rat, finely mincing and then treating with 0.5% trypsin
in EDTA. Cells were seeded in DMEM/F12 (Invitrogen) with 10% PBS
(Sigma) and 2 mM Glutamax on T75 flasks coated with poly-L-lysine
and shaken after 4 hours to remove non-adherent cells. Astrocytes
were matured in culture for at least 28 days. Astrocytes were
harvested with EDTA and a cell scraper and resuspended is
Neurobasal-A plus HEPES at a density of 5.0.times.10.sup.5/70 uL
for time-lapse microscopy experiments.
11. Cortical Microglia Preparation
[0183] Cortical microglia were collected by removing the cortices
of a P0-P1 rat, finely mincing and then treating with 0.5% trypsin
in EDTA. Cells were plated in DMEM/F12 (Invitrogen) with 20% FBS
(Sigma) and 2 mM Glutamax on T75 flasks coated with poly-L-lysine
for 5-7 days. One day prior to time-lapse experiments, flasks were
agitated to remove less adherent cells and these cells were plated
in uncoated pears dishes (Falcon) in D10F at a density of
1.0.times.10.sup.6/mL. The following day, the primary microglia
were harvested with EDTA and a cell scraper and resuspended in
Neurobasal-A plus HEPES at a density of 5.0.times.10.sup.5/70 uL
for time-lapse microscopy experiments.
12. Time-lapse Microscopy Studies
[0184] DRG neurons were incubated at 37.degree. C. for 48 hours
prior to time-lapse imaging. Neurobasal-A media with HEPES (50 uM,
Sigma) was added to the culture prior to transfer to a heated stage
apparatus. Time-lapse images were acquired every 30 seconds for 3
hours with a Zeiss Axiovert 405M microscope using a 100.times.
oil-immersion objective. Growth cones were chosen that extended
straight into the spot rim and had characteristic dystrophic
morphology. Neurons were observed for 30 minutes to determine
baseline behavior before the addition of additional cell types (N-6
for all groups except primary macrophage, N-3). Growth cones were
Observed for 150 minutes after cell addition. We tracked
extension/retraction, and rate of growth with Metamorph
software.
13. Statistical Analysis
[0185] Data were analyzed by One- or Two-way ANOVA or General
linear model, where appropriate, and Tukey post-hoc test with
Minitab 15 software.
Discussion
[0186] The results show for the first time conclusive evidence that
macrophages induce retraction of dystrophic adult axons through
direct physical contact. The induction of retraction was dependent
upon both the growth state of the neurons and the activation state
of the macrophages. Primary bone marrow-derived macrophages
required stimulation with interferon-gamma and LPS to reach a state
of activation similar to the cell line macrophages and macrophages
within a spinal cord lesion to induce axonal retraction in vitro.
This indicates that the behavior of macrophages in vivo is
state-dependent and corresponds with previous work showing that
macrophage infiltration only correlates with axonal retraction in
the presence of myelin degeneration (McPhail et al., 2004). The
study shows that adult sensory neurons were only susceptible to
macrophage-induced retraction when they were in a dystrophic state
of stalled growth induced by a gradient of inhibitory CSPG, Adult
neurons in an active state of growth on a uniform laminin substrate
rapidly broke contacts with macrophages and did not retract.
[0187] The induction of retraction potentially involves multiple
intrinsic and extrinsic mechanisms. The study shows that retraction
did not occur without direct cell-cell contact between macrophages
and dystrophic neurons. The addition of trypsin-treated macrophages
or macrophage-conditioned media alone was insufficient to induce
retraction. Macrophages did not have to contact dystrophic axons
specifically at the growth cone in order to induce retraction. It
is possible that contact with activated macrophages may trigger
signaling pathways within the axon distant from its dystrophic
ending.
[0188] There are several candidate binding partners by which
macrophages and neurons may physically identify to one another and
interact. Macrophages may use alphas and betal integrin receptors
to recognize and hind to axonal vitronectin (Sobel at al., 1995)
and macrophage adhesion to degenerating peripheral nerve is
partially attenuated by blocking betal integrin (Brown at al.,
1997). Following injury to the optic nerve, axons express ephrinB3,
which is recognized by the EphB3 receptor present on macrophages
(Liu at al., 2006). Sialoadhesin, a macrophage-specific receptor
for sialic acid, is present on neuronal cell membranes (Kelm et
al., 1994; Tang et al., 1997). Macrophages also recognize
phosphatidylserine exposed on the outer membrane surface of cells
undergoing apoptosis (De et al., 2002; De Simeone at al., 2004)
which may flag injured neurons for endocytosis. Additionally,
fractalkine is a chemokine expressed predominantly on the surface
of CNS neurons, while its receptor, CX3CR1, is found on macrophages
(Zujovic et al., 2000; Umehara at al., 20011:. Further studies must
be done to determine which, if any, of these molecules arc
expressed or upregulated on the surfaces of dystrophic adult
neurons targeting them for macrophage recognition.
[0189] This study confirms that the ascending dorsal column pathway
undergoes retraction (Horgens et al., 1986; McPhail et al., 2004;
Stirling et al., 2004; Baker and Hagg, 2005; Baker et al., 2007)
and the timing corresponds to the infiltration of macrophages.
Axonal retraction has been examined in other pathways within the
spinal cord including the descending cortical spinal tract (Fishman
and Kelley, 1954; Iizuka et al., 1957; Hill et al., 2001; Seif et
al., 2007), bulbospinal tract (Houle and Jin, 2001), and
rubrospinal tract (Schwartz et al., 2005; Cao at al., 2007). It is
important to consider that there are two distinct phases of axonal
retraction, A recent study which imaged axotomized ascending
sensory axons of the adult mouse in vivo showed axonal retraction
of about 300 um within the first few hours of injury followed by
axon stabilization for the first three days post-lesion
(Kerschensteiner et al., 2005). Therefore the focus of this study
was on the second, later phase of axonal retraction that is due to
activated macrophages and not to the intrinsic properties of the
neurons.
[0190] This study shows that treatment with clodronate liposomes
prior to injury and throughout the normal retraction period
prevented the second phase of axonal retraction between two and
seven days post-lesion. Previous work has shown that clodronate
liposome-mediated macrophage depletion results in reduced lesion
volume and increased neuronal survival (Popovich at al., 1999; van
Rooijen and van Kesteren-Hendrix, 2002). While these authors stress
the axon regenerative effect of macrophage depletion, the data in
this study suggest that the increased axonal content of the lesions
of clodronate liposome-treated animals may be due, at least in
part, to an attenuation of axonal retraction.
Example III
Stem Cells can Prevent Adhesion of Activated Macrophages to
DRGs
Results
1. Model
[0191] Following dorsal column crush injury, regenerating axons
encounter macrophages and microglia and form dystrophic endings.
This is shown schematically in FIG. 1. Previous work from the
inventors' laboratory has shown that macrophage infiltration is
correlated with axonal dieback following dorsal column crush injury
(FIGS. 2-3C). After characterizing the extent of axonal dieback of
the ascending dorsal column sensory axons following injury, the
inventors established an in vitro model of dieback, which can he
used to evaluate various treatment strategies. The in vitro assay
consists of cultured adult DRG neurons on a substrate of opposing
gradients of the growth-promoting protein laminin and the potently
inhibitory chondroitin sulfate proteoglycan aggrecan (Tom et al.,
2004). This spot gradient is sufficient to stall axonal growth and
induce the formation of dystrophic growth cones like those observed
in the injured spinal cord.
[0192] Time-lapse microscopy allowed the inventors to closely
examine growth cone dynamics, such as the number of filopodia,
extent of lamellapodia, and number of vesicles in the dystrophic
endings. Direct cell-cell contacts were frequently observed between
dystrophic axons and macrophages leading to extensive retraction of
the axon (FIGS. 4A-5D). Direct cell contact was necessary to induce
retraction, as neither macrophage-conditioned media, nor the
presence of macrophages near dystrophic axons resulted in
retraction. Therefore, the inventors hypothesized that depletion or
modulation of activated macrophages may be a potential therapeutic
target in spinal cord. injury.
[0193] The inventors have elucidated the mechanism by which
macrophage-neuron interactions result in dieback. Macrophages are
known to secrete a variety of proteases, which aid in the breakdown
and clearance of debris, and the inventors have shown that
macrophages express and secrete MMP-9. They hypothesized that a
protease could be responsible for locally dislodging a dystrophic
axon from the substrate causing it to retract. One class of
proteases expressed by macrophages is the matrix metalloproteinase
(MMP). MMPs have already been implicated in regeneration failure in
the CNS as transgenic mice lacking certain MMPs exhibit enhanced
axonal regrowth following injury, as do animals treated with the
general MMP inhibitor, GM6001. GM6001, which acts as a zinc
chelator at MMP active sites, was applied to the timelapse dish at
the time of macrophage addition. Treatment with GM6001 or a
specific MMP-9 inhibitor (FIGS. 6A-C) in the in vitro model
prevented the retraction of dystrophic growth cones after direct
cell-cell contact with macrophages, while a specific MMP-2
inhibitor did not, GM6001 and the specific MMP-9 inhibitor did not
prevent the direct cell-cell contact between macrophages and
dystrophic axons. Thus, MMPs, and MMP-9 are implicated as playing a
role in axonal dieback.
2. MAPCs Prevent Macrophage-Mediated Axonal Dieback in vitro
[0194] FIG. 7 shows a schematic representation of the experimental
design to determine if MAPCs could modulate the inhibitory effects
of macrophages, MAPCs were added to 1 DIV DRG spot cultures and
incubated for an additional day. Growth cone morphology of these
cocultured neurons was quite different from dystrophic growth cones
typically found on the spot. These growth cones were increasingly
motile, flattened and had extensive lamellapodia. Macrophages
contacted the growth cone and axon, but these contacts were often
transient, and 5 out of 6 axons imaged did not undergo the
characteristic macrophage-mediated retraction (FIGS. 8A-B).
[0195] This result could be to due to neurostimulatory or
immunomodulatory effects of the MAPCs, or both. To address this
question, a series of conditioned-media experiments were performed.
Dissociated DRG neurons were treated for 24 hours with
MAPC-conditioned media and compared with media controls to assess
the longest neurite. No significant difference was seen in the
length of neurites between groups, suggesting that the effect might
not be entirely neurostimulatory.
[0196] Direct addition of MAPC-conditioned media to the timelapse
dish resulted in a change in growth cone morphology, from a
dystrophic, stalled state, to a motile, flattened state.
Macrophages still contacted these axons, but contacts were
generally transient and generally did not result in axonal
retraction. Macrophages pretreated with MAPC-conditioned media also
contacted axons on the spot, but did not cause retraction (FIGS.
9A-12). It is possible that MAPCs act on macrophages to alter their
receptor expression, response to injured cells, or secretion of
MMP-9.
3. MAPCs Decrease the Extent of Axonal Dieback Following Dorsal
Column Crush Injury
[0197] The immune-modulating effect of MAPCs on axonal dieback in
vivo was investigated using a dorsal column crush model of spinal
cord injury. The most dramatic phase of axonal dieback. occurs
between two and four days post-lesion, which correlates
spatiotemporally with the infiltration of activated macrophages
into the lesion. It was possible that MAPCs would modulate
activated macrophages within the lesion in such a way as to reduce
the amount of axonal dieback, Therefore, MAPCs were transplanted
into the spinal cord immediately following injury and the extent of
axonal dieback was measured at two and four days post-lesion. The
MAPCs were transplanted approximately 500 microns caudal to the
lesion and 500 microns lateral to the midline. This location was
chosen in order to place the MAPCs close to the ends of the injured
axons, to further disruption of the ascending tract, and to prevent
the cells from being displaced from the spinal cord by blood and
CSF flow directly at the lesion site.
[0198] The transplanted MAPCs successfully integrated into the
spinal cord tissue as was evidenced by the presence of GFP.sup.+
cells at the injection site at both two and four days post-lesion.
In addition, the MAPCs migrated extensively away from the site of
transplantation and were able to occupy the core of the lesion and
were also observed to associate with the endings of injured axons.
At two days post-lesion, the extent of axonal dieback in MAPC
transplanted animals not significant from that of control animals
(FIGS. 13A-B). MAPC transplantation did not prevent the extent of
axonal tieback normally observed at two days post-lesion. However,
this initial phase of tieback is most likely due to intrinsic
neuronal mechanisms and is not mediated by activated macrophages,
they have not yet infiltrated the lesion at this time.
[0199] At four days post-lesion, MAPC transplanted animals showed a
significant decrease in the extent of axonal tieback compared to
non-injected controls (FIG. 13). The transplantation of MAPCs
nearly completely attenuated the dieback normally observed at this
time, which this study has shown to be directly caused by the
infiltration of activated macrophages, Therefore, the presence of
MAPCs within the injured spinal cord is sufficient to reduce
macrophage-induced axonal dieback in vivo.
Example IV
[0200] Vimentin/NG2.sup.+ oligodendrocyte precursor cells in the
lesion core start to expand around the time of macrophage
infiltration, and the ends of axotomized fibers are associated with
this cell population. This suggested that NG2.sup.+ cells within a
CNS lesion serve to stabilize axons, making them an ideal candidate
to prevent macrophage-mediated retraction. NG2.sup.+ glial cells
from adult mouse spinal cord were added to DRG cultures after one
day in vitro. On day 2, following a 30-minute period of baseline
observation, NR 8383 macrophages were added to the timelapse dish
and observed for 2.5 additional hours. The presence of NG2.sup.+
glial cells in coculture with DRGs was not sufficient to prevent
macrophage-induced axonal retraction (N=5). in FIG. 14A-C, the axon
retracts following macrophage contact and stabilizes on an
NG2.sup.+ glial cell.
Methods
1. DRG Dissociation
[0201] DRGs were harvested as previously described (Tom et al.,
2004; Davies et al,, 1999). Briefly, DRGs were dissected out of
adult female Sprague-Dawley rats (Harlan). Both the central and
peripheral roots were removed and ganglia. incubated in a solution
of Collagenase II (200 U/mL, Worthington) and Dispase II (2.5 U/mL,
Roche) in HBSS. The digested DRGs were rinsed and gently triturated
in fresh HBSS-CMF three times followed by low speed centrifugation.
The dissociated DRGs were then resuspended in Neurobasal-A media
supplemented with B-27, Glutamax, and Pennicillin/Streptamycin (all
from Invitrogen) and counted. DRGs were plated on Delta-T dishes
(Fisher) at a density of 3,000 cells/mL for a total of 6,000
cells/dish.
2. Timelapse Dish Preparation
[0202] Delta-T cell culture dishes (Fisher) were prepared similarly
to Tom et al., 2004. Briefly, a single hole was drilled through the
upper half of each dish with a number 2 bit to create a port for
the addition of cells to the cultures during timelapse microscopy.
Dishes were then rinsed with sterile water and coated with
poly-1-lysine (0.1 mg/mL, Invitrogen) overnight at room
temperature. Dishes were then rinsed with sterile water and allowed
to dry. Aggrecan gradient spots were created by pipetting 2.0 uL of
aggrecan solution (2.0 mg/mL, Sigma in HBSS-CMF, Invitrogen) onto
the culture surface and allowed to dry. Six spots were placed per
dish. After the aggrecan spots dried completely, the entire surface
of the dish was bathed in laminin solution (10 ug/mL, BTI in
HBSS-CMF) for three hours at 37 degrees Celsius. The laminin bath
was then removed immediately before plating of cells.
3. Cell Line Macrophage Cultures
[0203] NR8383 cells (ATCC # CRL-2192), an adult Sprague-Dawley
alveolar macrophage cell line, were cultured as described in Yin et
al., 2003. Briefly, cells were cultured in uncoated tissue culture
flasks (Corning) in F-12K media (Invitrogen) supplemented with 15%
FBS (Sigma), Glutamax, Penn/Strep (Invitrogen), and sodium
bicarbonate (Sigma) and fed two to three times per week. To prepare
the cell line macrophages for timelapse microscopy experiments,
cells were harvested with trypsin/EDTA (Invitrogen), washed three
times, and plated in uncoated tissue culture flasks at a density of
1.0.times.10.sup.6/mL in serum-free F-12K. Prior to use in
timelapse experiments, the cultured cell line magrophages were
harvested with EDTA and a cell scraper and resuspended in
Neurobasal-A with the addition of HEPES (50 uM, Sigma) at a density
of 2.5.times.10.sup.5/70 ul.
4. MAPC Cultures
[0204] Sprague-Dawley rat MAPC labeled with OFF were grown in rat
MAPC media consisting of low glucose DMEM (Invitrogen)
0.4.times.MCDB-201 medium (Sigma). 1.times. ITS liquid media
supplement (Sigma), 1 mg/ml linoleic acid-albumin (Sigma), 100 U/ml
sodium/100 .mu.g/ml streptomycin sulfate (Invitrogen), 100 .mu.M
2-P-L-ascorbid acid (Sigma), 100 ng/ml EGF (Sigma), 100 ng/ml PDGF
(R&D Systems), 50 dexamethasone (Sigma), 1000 U/ml ESGRO
(Chemicon), and 2% fetal bovine serum (Hyclone). The cultures were
plated on 10 ng/ml fibronectin (Invitrogen) coated 150 cm.sup.2
tissue culture flasks (Corning) at an initial density of 1000
cell/cm2 and subsequent replating at 200 cells/cm.sup.2. The cells
were maintained in 15 ml of media/flask at 37.degree. C. and 5.0%
CO.sub.2 with passaging occurring every 3-4 days using trypsin/EDTA
(Invitrogen).
5. MAPC-Conditioned Media
[0205] Cells were cultured as described above and conditioned media
was collected after 48 hours in 50 ml conical tubes (BD
Bioscience). The conditioned media was spun down at 400.times.g for
5 min at 4.degree. C. and the supernatant transferral to a new 50
ml conical tube. The conditioned media was that stored at 4.degree.
C.
[0206] MAPC-conditioned media was obtained as described above and
concentrated 50 fold with an Amicon Microcon Ultracel YM-3 3,000
MWCO centrifugal filter (Millipore, Bedford MA).
6. MAPC-Conditioned Media-Treated Macrophage
[0207] NR8383 rat macrophages were cultured as described above. One
day prior to timelapse microscopy experiments, macrophages were
harvested with trypsin/EDTA (Invitrogen), washed three times, and
plated in uncoated tissue culture flasks at a density of
1.0.times.10.sup.6/mL in serum-free F-12K. Twenty uL of the 50-fold
concentrated MAPC-conditioned media were added per 1 mL of
serum-free F12K media, for a final concentration of 1.times.. Prior
to use in timelapse experiments, the cultured cell line macrophages
were harvested with EDTA and a cell scraper and resuspended in
Neurobasal-A with the addition of HEPES (50 uM, Sigma) at a density
of 2.5.times.10.sup.5/70 ul.
7. Timelapse Microscopy
[0208] DRG neurons were incubated at 37.degree. C. for 48 hours
prior to timelapse imaging. Neurobasal-A media with HEPES (50uM,
Sigma) was added to the culture prior to transfer to a heated stage
apparatus. Time-lapse images were acquired every 30 seconds for 3
hours with a Zeiss Axiovert 405M microscope using a 100.times.
oil-immersion objective. Growth cones were chosen that extended
straight into the spot rim and had characteristic dystrophic
morphology for 30 minutes to observe baseline behavior before the
addition of cells or conditioned media and then observed for 3
hours.
[0209] For cell-addition experiments, cultured rat-derived MAPCs
were harvested from tissue culture flasks, washed three times and
resuspended in Neurobasal-A media. For coculture experiments, MAPCs
(100,000/dish) were added to dorsal root ganglia neuron cultures
after 24 hours and incubated at 37.degree. C. for 24 additional
hours before timelapse imaging.
[0210] For experiments in which MAPC-conditioned media was added to
DRG cultures during timelapse imaging, 90 uL of 50.times. MAPC-CM
was added after 30 minutes of baseline growth cone observation.
[0211] MAPC-conditioned media-treated macrophages were added to
timelapse cultures after 30 minutes of baseline imaging (500,000
cells/dish),
[0212] Extension/retraction, rate of growth, turning and branching
were analyzed using Metamorph software.
8. Immunocytochemisty
[0213] Following timelapse imaging, DRGs were fixed in 4% PFA and
immunostained with anti-B-tubulin-type III (1:500; Sigma),
anti-chondroitin sulfate (CS-56, 1:500, Sigma) and anti-GFP (1:500,
Invitrogen).
9. Primary Bone Marrow-Derived Macrophage Cultures
[0214] Bone marrow progenitor cells were harvested as described in
Tobian et al. 2004. Briefly, femurs were removed from adult female
Sprague-Dawley rats (Harlan). The ends of the femurs. were removed,
a syringe containing cold DMEM supplemented with 10% FBS, Glutamax,
Penn/Strep, beta-mercaptoethanol, and HEPES (Invitrogen) (D10F) is
inserted into the femur and the bone marrow was flushed out and
collected. The resulting cell mixture was then passed through a 70
um filter and centrifuged. Supernatant was then removed, the
resulting cell pellet resuspended in AKT lysing buffer
(BioWhitacre) to lyse red blood cells, and centrifuged. The
supernatant was removed and the pellet containing bone marrow
precursor cells was resuspended and plated in DMEM above
additionally supplemented with 20% LADMAC cell line-conditioned
media (Generous gift of Dr. Clifford Harding) to induce
differentiation into macrophages. Cells were harvested for
experimentation on day 10 in culture. One day prior to timelapse
experiments, primary macrophages were harvested with trypsin/EDTA,
washed three times with D10F, and plated in uncoated petri dishes
(Falcon) in D10F at a density of 1.0.times.10.sup.6/ml. The
following day, the primary macrophages were harvested with EDTA and
a cell scraper and resuspended in Neurobasal-A plus HEPES at a
density of 5.0.times.10.sup.5/70 ul for timelapse microscopy
experiments.
10. Dorsal Column Crush Lesion Model
[0215] Adult Female Sprague-Dawley rats 250-300 g were anesthetized
with inhaled isofluorane gas (2%) for all surgical procedures. A T1
laminectomy was performed to expose the dorsal aspect of the C8
spinal cord segment. A durotomy was made bilaterally 0.75 mm from
midline with a 30 gauge needle. A dorsal column crush lesion was
then made by inserting Dumont # 3 jeweler's forceps into the dorsal
spinal cord at C8 to a depth of 1.0 mm; squeezing the forceps
holding pressure for ten seconds and repeated two additional times.
Completion of the lesion was verified by observation of white
matter clearing, The holes in the dura were then covered with gel
film. The muscle layers were sutured with 4-0 nylon suture and the
skin closed with surgical staples. Upon closing of the incision,
animals received Marcaine (1.0 mg/kg) subcutaneously along the
incision as well as Buprenorphine (0.1 mg/kg) intramuscularly.
Post-operatively, animals were kept warm with a heating lamp during
recovery from anesthesia and allowed access to food and water ad
libitum. Animals were killed at 2, 4, 7, 14, or 28 days
post-lesion.
11. Cell Transplantation
[0216] Cultured rat-derived MAPCs or primary bone marrow-derived
macrophages (which had been stimulated with interferon-gamma and
LPS for 24 hours) were harvested from tissue culture flasks, washed
three times in HBSS-CMF and resuspended in HBSS-CMF at a density of
200,000 cells/uL. Immediately following dorsal column crush injury,
1.0 uL of the cell suspension was injected unilaterally 0.5 mm deep
into the right side dorsal columns. The injection site was 0.5 mm
lateral to the midline and 0.5 mm caudal to the lesion edge. The
cells were injected with forty-four 23.0 nL pulses on 15 second
intervals through a pulled glass pipette attached to a Nanoject II
(Drummond). The glass pipette was then withdrawn from the spinal
cord two minutes after the final injection. Following the
transplantation, the injection site was covered with gelfilm, the
muscle layers were closed with 4-0 ethicon sutures, and the skin
was closed with surgical staples. Post-operatively, animals were
kept warm with a heating lamp during recovery from anesthesia and
allowed access to food and water ad libitum. Animals were killed
two or four days post-lesion.
12. Axon Labeling
[0217] Two days before sacrifice, the dorsal columns were labeled
unilaterally with Texas-Red conjugated 3000 MW dextran. Briefly,
the sciatic nerve of the right hindlimb was exposed and crushed
three times with Dumont #3 forceps for ten seconds. 1.0 uL of 3000
MW dextran-Texas-Red 10% in sterile water was the injected via a
Hamilton syringe into the sciatic nerve at the crush site.
[0218] The muscle layers were closed with 4-0 nylon suture and the
skin with surgical staples. Upon closing of the incision, animals
received Marcaine (1.0 mg/kg) subcutaneously along the incision as
well as Buprenorphine (0.1 mg/kg) intramuscularly.
Post-operatively, animals will be kept warm with a heating lamp
during recovery from anesthesia and allowed access to food and
water ad libitum. Animals were killed two days following labeling
with an overdose of isofluorane and perfused with PBS followed by
4% PFA. Tissue was harvested and post-fixed in 4% HA and processed
for immunohistochemistry.
13. Immunohistochemistry
[0219] Tissue was post-fixed in 4% PFA overnight and then submersed
in 30% sucrose overnight frozen in OCT mounting media, and cut on a
cryostat into 20 um longitudinal sections. Tissue was then stained
with anti-GFAP/Alexafluor-405, anti-ED-1/Alexafluor-594 or -633,
anti-GFP/Alexafluor-488, and anti-vimentin/Alexafluor-533. And then
imaged on a Zeiss Axiovert 510 laser-scanning confocal microscope
at 10.times. magnification.
14. Axonal Dieback Quantification
[0220] To quantify axonal dieback three consecutive sections per
animal were analyzed, starting at a depth of 200 um below the
dorsal surface of the spinal cord. The lesion center was identified
via characteristic GFAP and/or vimentin staining patterns and then
centered using Zeiss LSM 5 Image Browser software. The distance
between the ends of 5 labeled axons projecting farthest into the
lesion and the lesion center was then pleasured. The measurements
from all sections from all animals in a group were averaged to
yield the average distance of dieback per time point.
Example V
[0221] Mesenchymal stem cells can he commercial obtained. For
example, Rat Mesenchymal Stem Cell Kit (Millipore Catalog No.
SCR026) provides read-to-use primary mesenchymal stem cells
isolated from the bone marrow of adult Fisher 344 rats along with a
panel of positive and negative selection markers for the
characterization of mesenchymal stem cell population. Positive cell
markers include antibodies directed again two cell-surface
molecules (integrin b1 and CD54) that are present on mesenchymal
stem cells. Negative cell markers include antibodies directed
against two specific hematopoietic cell surface markers, (CD14,
present on leukocytes and CD45, present on monocytes and
macrophages) that are not expressed by mesenchymal stem cells.
These mesenchymal stem cells were assessed for the ability to
reduce retraction and were found to reduce retraction (reduce
adhesion) in vitro (glial scar).
Example VI
[0222] MAPC-conditioned media treatment of adult DRGs grown on 5
.mu.g/ml laminin promotes neurite outgrowth. See FIG. 16. The
longest axon from each dissociated DRG was measured for the group
to Which media containing Neurobasal-A and either MAPC-conditioned
media, control media, or no additional media were added. All
conditions are significant from one another, One-way ANOVA,
*p<0.0001. B, 16.times. image representing the average amount of
outgrowth of an untreated DRG neuron. C, 16.times. image
representing the average amount of outgrowth of DRGs pretreated
with MAPC-conditioned media.
[0223] This result could be to due to neurostimulatory or
immunomodulatory effects of the MAPCs, or both. To address this
question, a series of conditioned-media experiments ware performed.
Dissociated DRG neurons were treated for 24 hours with fresh
MAPC-conditioned media and compared with media controls. The
longest neurite of each DRG was measured. Fresh MAPC-conditioned
media significantly increased outgrowth on laminin. This suggests
that MAPCs secrete one or more growth factors that have a
neurostimulatory effect on adult neurons. Previously, frozen
MAPC-conditioned media did not have the same effect on neurons,
suggesting that the factor was altered or inactivated in the
freezing process.
REFERENCES
[0224] Arantes et al., Neuroreport 11:177-181 (2000)
[0225] Baker et al., Exp Neurol 205:82-91 (2007)
[0226] Baker and Flagg, J Neurotrauma 22:415-428 (2005)
[0227] Basch at al., (1983)
[0228] Batinic, D., et al., (1990)
[0229] Borgens et al., J Comp Neurol 250:157-167 (1986)
[0230] Brown et al., Eur Neurosci 9:2057-2063 (1997)
[0231] Busch and Silver, Curr Opin Neuobiol 17:120-127 (2007)
[0232] Cai et al., (1988)
[0233] Cao el al., Neurorehabil Neural Repair (2007)
[0234] Chang, P., et al., (1999)
[0235] Chang, T. M., (1992)
[0236] Davies et al., J Neurosci 19:5810-5822 (1999)
[0237] De et al., J Neuropathol Exp Neurol 61:237-244 (2002)
[0238] De Simone et al., Mol Neurobio 29:197-212 (2004)
[0239] Donnelly and Popovich, Exp Neural 209:378-388 (2008)
[0240] Farr et al., Aplysia J Neurobiol 46:89-96 (2001)
[0241] Fayaz and Tator, J Neurosci Methods 102:69-79 (2000)
[0242] Fishman and Kelley, Neurology 34:1161-1167 (1984)
[0243] Fitch and Silver Exp Neurol 148:587-603(1997)
[0244] Fitch et al., J Neurosci 19:8182-8198 (1999)
[0245] Grimpe and Silver, J Neurosci 24:1393-1397 (2004)
[0246] Hill et al., Exp Neurol 171:153-469 (2001)
[0247] Hohlfeld et al., Neurology 68:S58-S63 (2007)
[0248] Houle and Jin, Exp Neurol 169:208-217 (2001)
[0249] Iizuka et al. J Neurosurg 66:595-603 (1987)
[0250] Jones et al., Exp Neurol 182:399-411 (2003)
[0251] Kelm et al., Curr Biol 4:965-972 (1994)
[0252] Kerschensteiner et al., Nat Med 11:572-577 (2005)
[0253] Kwon et al., Proc Natl Acad Sci USA 99:3246-3251 (2002)
[0254] Li and Raisman, Exp Neural 134:102-111 (1995)
[0255] Liu et al., J Neurosci 26:3087-3101 (2006)
[0256] Longbrake et al., Neurochem 102:1083-1094 (2007)
[0257] Matthew, et al., (1991)
[0258] McKeon et al., J Neurosci 11:3398-3411 (1991)
[0259] McPhail et al., Eur J. Neurosci 20:19844994 (2004)
[0260] Morgenstern et al., Prog Brain Res 137:313-322 (2002)
[0261] Muschler, G. F., et al., (1997)
[0262] Oudega et al., Eur J. Neurosci 11:2453-2464 (1999)
[0263] Prewitt et al., Exp Neurol 148:433-443 (1997)
[0264] Popovich et al., J Comp Neurol 377:443-464 (1997)
[0265] Popovich and Longbrake, Nat Rev Neurosci 9:481-493
(2008)
[0266] Popovich et al., Exp Neurol 158:351-365 (1999)
[0267] Raff et al., Science 296:868-871 (2002)
[0268] Ramon y Cajal, London: Oxford UP (1928)
[0269] Rapalino et al., Nat Med 4:814-821 (1998)
[0270] Schwartz et al. Neurosurgery 44:1041-1045 (1999b)
[0271] Schwartz et al., Am J Neuroradial 267-18 (2005)
[0272] Seif et al., J. Neurotrauma 24:1513-1528 (2007)
[0273] Silver and Miller, Nat Rev Neurosci 5: 146-156 (2004)
[0274] Snow et al., Exp Neurol 109:111-130 (1990)
[0275] Sobei et al., Exp Neurol 54:202-213 (1995)
[0276] Steinmetz et al., Neurosci 25:8066-8076 (2005)
[0277] Stirling et al, J Neurosci 24:2182-2190 (2004)
[0278] Tang et al., J Neurosci Res 71;427-444 (2003)
[0279] Tang et al., J Cell Biol 138:1355-1366 (1997)
[0280] Tobian et al., J Immunol 172:5277-5286 (2004)
[0281] Tom et al. J Neurosci 24:6531-6539 (2004)
[0282] Umehara et al., Immunol Cell Biol 79:298-302 (2001)
[0283] van Rooijen et al., Trends Biotechnol 15:178-185 (1997)
[0284] van Rooijen and van Kesteren-Hendrikx, J Liposome Res
12:81-94 (2002)
[0285] van Rooijen and Sanders, J Immuno Methods 174:83-93
(1994)
[0286] Watanabe et al., J Neurotrauma 16:255-265 (1999)
[0287] Wysocki and Sato, (1978)
[0288] Xie and Zheng, Exp Neurol 209:302-312 (2008)
[0289] Yanagi, K. et al., (1939)
[0290] Yin et. al., J Neurosci 23:2284-2293 (2003)
[0291] Yiu and He, Nat Rev Neurosci 7:617-627 (2006)
[0292] Zujovic et al., Glia 29:305-315 (2000)
* * * * *